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2009
Olieric V, Rieder U, Lang K, Serganov A, Schulze-Briese C, Micura R, Dumas P, Ennifar E
A fast selenium derivatization strategy for crystallization and phasing of RNA structures Article de journal
Dans: RNA, vol. 15, no. 4, p. 707-715, 2009, ISBN: 19228585, (1469-9001 (Electronic) Journal Article Research Support, Non-U.S. Gov't Validation Studies).
Résumé | Liens | BibTeX | Étiquettes: Base Sequence Crystallography, ENNIFAR, Unité ARN, X-Ray/*methods HIV-1/chemistry/genetics Nucleic Acid Conformation RNA/*chemistry Selenium/*metabolism Staining and Labeling/*methods
@article{,
title = {A fast selenium derivatization strategy for crystallization and phasing of RNA structures},
author = {V Olieric and U Rieder and K Lang and A Serganov and C Schulze-Briese and R Micura and P Dumas and E Ennifar},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19228585},
isbn = {19228585},
year = {2009},
date = {2009-01-01},
journal = {RNA},
volume = {15},
number = {4},
pages = {707-715},
abstract = {Site-specific 2'-methylseleno RNA labeling is a promising tool for tackling the phase problem in RNA crystallography. We have developed an efficient strategy for crystallization and structure determination of RNA and RNA/protein complexes based on preliminary crystallization screening of 2'-OCH(3)-modified RNA sequences, prior to the replacement of 2'-OCH(3) groups with their 2'-SeCH(3) counterparts. The method exploits the similar crystallization properties of 2'-OCH(3)- and 2'-SeCH(3)-modified RNAs and has been successfully validated for two test cases. In addition, our data show that 2'-SeCH(3)-modified RNA have an increased resistance to X-ray radiolysis in comparison with commonly used 5-halogen-modified RNA, which permits collection of experimental electron density maps of remarkable quality.},
note = {1469-9001 (Electronic)
Journal Article
Research Support, Non-U.S. Gov't
Validation Studies},
keywords = {Base Sequence Crystallography, ENNIFAR, Unité ARN, X-Ray/*methods HIV-1/chemistry/genetics Nucleic Acid Conformation RNA/*chemistry Selenium/*metabolism Staining and Labeling/*methods},
pubstate = {published},
tppubtype = {article}
}
Site-specific 2'-methylseleno RNA labeling is a promising tool for tackling the phase problem in RNA crystallography. We have developed an efficient strategy for crystallization and structure determination of RNA and RNA/protein complexes based on preliminary crystallization screening of 2'-OCH(3)-modified RNA sequences, prior to the replacement of 2'-OCH(3) groups with their 2'-SeCH(3) counterparts. The method exploits the similar crystallization properties of 2'-OCH(3)- and 2'-SeCH(3)-modified RNAs and has been successfully validated for two test cases. In addition, our data show that 2'-SeCH(3)-modified RNA have an increased resistance to X-ray radiolysis in comparison with commonly used 5-halogen-modified RNA, which permits collection of experimental electron density maps of remarkable quality.
Olieric V, Wolff P, Takeuchi A, Bec G, Birck C, Vitorino M, Kieffer B, Beniaminov A, Cavigiolio G, Theil E, Allmang C, Krol A, Dumas P
SECIS-binding protein 2, a key player in selenoprotein synthesis, is an intrinsically disordered protein Article de journal
Dans: Biochimie, vol. 91, no. 8, p. 1003-1009, 2009, ISBN: 19467292, (1638-6183 (Electronic) Journal Article Research Support, Non-U.S. Gov't).
Résumé | Liens | BibTeX | Étiquettes: ENNIFAR, ERIANI, Unité ARN
@article{,
title = {SECIS-binding protein 2, a key player in selenoprotein synthesis, is an intrinsically disordered protein},
author = {V Olieric and P Wolff and A Takeuchi and G Bec and C Birck and M Vitorino and B Kieffer and A Beniaminov and G Cavigiolio and E Theil and C Allmang and A Krol and P Dumas},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19467292},
isbn = {19467292},
year = {2009},
date = {2009-01-01},
journal = {Biochimie},
volume = {91},
number = {8},
pages = {1003-1009},
abstract = {Selenocysteine (Sec) is co-translationally incorporated into selenoproteins at a reprogrammed UGA codon. In mammals, this requires a dedicated machinery comprising a stem-loop structure in the 3' UTR RNA (the SECIS element) and the specific SECIS Binding Protein 2. In this report, disorder-prediction methods and several biophysical techniques showed that ca. 70% of the SBP2 sequence is disordered, whereas the RNA binding domain appears to be folded and functional. These results are consistent with a recent report on the role of the Hsp90 chaperone for the folding of SBP2 and other functionally unrelated proteins bearing an RNA binding domain homologous to SBP2.},
note = {1638-6183 (Electronic)
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {ENNIFAR, ERIANI, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Selenocysteine (Sec) is co-translationally incorporated into selenoproteins at a reprogrammed UGA codon. In mammals, this requires a dedicated machinery comprising a stem-loop structure in the 3' UTR RNA (the SECIS element) and the specific SECIS Binding Protein 2. In this report, disorder-prediction methods and several biophysical techniques showed that ca. 70% of the SBP2 sequence is disordered, whereas the RNA binding domain appears to be folded and functional. These results are consistent with a recent report on the role of the Hsp90 chaperone for the folding of SBP2 and other functionally unrelated proteins bearing an RNA binding domain homologous to SBP2.
Wagner J, Etienne H, Fuchs R P, Cordonnier A, Burnouf D
Distinct beta-clamp interactions govern the activities of the Y family PolIV DNA polymerase Article de journal
Dans: Mol Microbiol, vol. 74, no. 5, p. 1143-1151, 2009, ISBN: 19843218, (1365-2958 (Electronic) 0950-382X (Linking) Journal Article).
Résumé | Liens | BibTeX | Étiquettes: DUMAS, ENNIFAR, Unité ARN
@article{,
title = {Distinct beta-clamp interactions govern the activities of the Y family PolIV DNA polymerase},
author = {J Wagner and H Etienne and R P Fuchs and A Cordonnier and D Burnouf},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19843218},
isbn = {19843218},
year = {2009},
date = {2009-01-01},
journal = {Mol Microbiol},
volume = {74},
number = {5},
pages = {1143-1151},
abstract = {The prototypic Y family DNA polymerase IV (PolIV) of Escherichia coli is involved in multiple replication-associated processes including spontaneous mutagenesis, translesion synthesis (TLS), cell fitness, survival under stressful conditions and checkpoint like functions. It interacts physically and functionally with the replisome's beta processivity clamp through the canonical PolIV C-terminal peptide (CTP). A second interaction that involves a portion of the little finger (LF) domain of PolIV has been structurally described. Here we show that the LF-beta interaction stabilizes the clamp-polymerase complex in vitro and is necessary for the access of PolIV to ongoing replication forks in vivo. However, in contrast to the CTP-beta, the LF-beta interaction is dispensable for the role of the polymerase in TLS. This discloses two independent modes of action for PolIV and, in turn, uncovers a novel way by which the cell may regulate the potentially deleterious effect of such low fidelity polymerases during replication.},
note = {1365-2958 (Electronic)
0950-382X (Linking)
Journal Article},
keywords = {DUMAS, ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The prototypic Y family DNA polymerase IV (PolIV) of Escherichia coli is involved in multiple replication-associated processes including spontaneous mutagenesis, translesion synthesis (TLS), cell fitness, survival under stressful conditions and checkpoint like functions. It interacts physically and functionally with the replisome's beta processivity clamp through the canonical PolIV C-terminal peptide (CTP). A second interaction that involves a portion of the little finger (LF) domain of PolIV has been structurally described. Here we show that the LF-beta interaction stabilizes the clamp-polymerase complex in vitro and is necessary for the access of PolIV to ongoing replication forks in vivo. However, in contrast to the CTP-beta, the LF-beta interaction is dispensable for the role of the polymerase in TLS. This discloses two independent modes of action for PolIV and, in turn, uncovers a novel way by which the cell may regulate the potentially deleterious effect of such low fidelity polymerases during replication.
Burnouf D Y, Wagner J E
Kinetics of deoxy-CTP incorporation opposite a dG-C8-N-2-aminofluorene adduct by a high-fidelity DNA polymerase Article de journal
Dans: J Mol Biol, vol. 386, no. 4, p. 951-961, 2009, ISBN: 19150355, (1089-8638 (Electronic) Journal Article Research Support, Non-U.S. Gov't).
Résumé | Liens | BibTeX | Étiquettes: ENNIFAR, Unité ARN
@article{,
title = {Kinetics of deoxy-CTP incorporation opposite a dG-C8-N-2-aminofluorene adduct by a high-fidelity DNA polymerase},
author = {D Y Burnouf and J E Wagner},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19150355},
isbn = {19150355},
year = {2009},
date = {2009-01-01},
journal = {J Mol Biol},
volume = {386},
number = {4},
pages = {951-961},
abstract = {The model carcinogen N-2-acetylaminofluorene covalently binds to the C8 position of guanine to form two adducts, the N-(2'-deoxyguanosine-8-yl)-aminofluorene (G-AF) and the N-2-(2'-deoxyguanosine-8-yl)-acetylaminofluorene (G-AAF). Although they are chemically closely related, their biological effects are strongly different and they are processed by different damage tolerance pathways. G-AF is bypassed by replicative and high-fidelity polymerases, while specialized polymerases ensure synthesis past of G-AAF. We used the DNA polymerase I fragment of a Bacillus stearothermophilus strain as a model for a high-fidelity polymerase to study the kinetics of incorporation of deoxy-CTP (dCTP) opposite a single G-AF. Pre-steady-state kinetic experiments revealed a drastic reduction in dCTP incorporation performed by the G-AF-modified ternary complex. Two populations of these ternary complexes were identified: (i) a minor productive fraction (20%) that readily incorporates dCTP opposite the G-AF adduct with a rate similar to that measured for the adduct-free ternary complexes and (ii) a major fraction of unproductive complexes (80%) that slowly evolve into productive ones. In the light of structural data, we suggest that this slow rate reflects the translocation of the modified base within the active site, from the pre-insertion site into the insertion site. By making this translocation rate limiting, the G-AF lesion reveals a novel kinetic step occurring after dNTP binding and before chemistry.},
note = {1089-8638 (Electronic)
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The model carcinogen N-2-acetylaminofluorene covalently binds to the C8 position of guanine to form two adducts, the N-(2'-deoxyguanosine-8-yl)-aminofluorene (G-AF) and the N-2-(2'-deoxyguanosine-8-yl)-acetylaminofluorene (G-AAF). Although they are chemically closely related, their biological effects are strongly different and they are processed by different damage tolerance pathways. G-AF is bypassed by replicative and high-fidelity polymerases, while specialized polymerases ensure synthesis past of G-AAF. We used the DNA polymerase I fragment of a Bacillus stearothermophilus strain as a model for a high-fidelity polymerase to study the kinetics of incorporation of deoxy-CTP (dCTP) opposite a single G-AF. Pre-steady-state kinetic experiments revealed a drastic reduction in dCTP incorporation performed by the G-AF-modified ternary complex. Two populations of these ternary complexes were identified: (i) a minor productive fraction (20%) that readily incorporates dCTP opposite the G-AF adduct with a rate similar to that measured for the adduct-free ternary complexes and (ii) a major fraction of unproductive complexes (80%) that slowly evolve into productive ones. In the light of structural data, we suggest that this slow rate reflects the translocation of the modified base within the active site, from the pre-insertion site into the insertion site. By making this translocation rate limiting, the G-AF lesion reveals a novel kinetic step occurring after dNTP binding and before chemistry.
2008
Lemay J, Maidou-Peindara P, Cancio R, Ennifar E, Coadou G, Maga G, Rain J C, Benarous R, Liu L X
AKAP149 binds to HIV-1 reverse transcriptase and is involved in the reverse transcription Article de journal
Dans: J Mol Biol, vol. 383, no. 4, p. 783-796, 2008, ISBN: 18786546, (1089-8638 (Electronic) Journal Article Research Support, Non-U.S. Gov't).
Résumé | Liens | BibTeX | Étiquettes: DUMAS A Kinase Anchor Proteins/chemistry/genetics/*metabolism Amino Acid Sequence Animals Binding Sites Cell Line HIV Reverse Transcriptase/chemistry/genetics/*metabolism HIV-1/enzymology/genetics Humans Models, ENNIFAR, Molecular Molecular Sequence Data Mutagenesis Protein Binding Protein Structure, Tertiary RNA Interference Recombinant Fusion Proteins/genetics/metabolism Reverse Transcription/*physiology Two-Hybrid System Techniques Virion/metabolism Virus Replication/physiology, Unité ARN
@article{,
title = {AKAP149 binds to HIV-1 reverse transcriptase and is involved in the reverse transcription},
author = {J Lemay and P Maidou-Peindara and R Cancio and E Ennifar and G Coadou and G Maga and J C Rain and R Benarous and L X Liu},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18786546},
isbn = {18786546},
year = {2008},
date = {2008-01-01},
journal = {J Mol Biol},
volume = {383},
number = {4},
pages = {783-796},
abstract = {Like all retroviruses, human immunodeficiency virus type 1 (HIV-1) undergoes reverse transcription during its replication cycle. The cellular cofactors potentially involved in this process still remain to be identified. We show here that A-kinase anchoring protein 149 (AKAP149) interacts with HIV-1 reverse transcriptase (RT) in both the yeast two-hybrid system and human cells. The AKAP149 binding site has been mapped to the RNase H domain of HIV-1 RT. AKAP149 silencing by RNA interference in HIV-1-infected cells inhibited viral replication at the reverse transcription step. We selected single-point mutants of RT defective for AKAP149 binding and demonstrated that mutant G462R, despite retaining significant intrinsic RT activity in vitro, failed to carry out HIV-1 reverse transcription correctly in infected cells. This suggests that the interaction between RT and AKAP149 in infected cells may play an important role in HIV-1 reverse transcription.},
note = {1089-8638 (Electronic)
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {DUMAS A Kinase Anchor Proteins/chemistry/genetics/*metabolism Amino Acid Sequence Animals Binding Sites Cell Line HIV Reverse Transcriptase/chemistry/genetics/*metabolism HIV-1/enzymology/genetics Humans Models, ENNIFAR, Molecular Molecular Sequence Data Mutagenesis Protein Binding Protein Structure, Tertiary RNA Interference Recombinant Fusion Proteins/genetics/metabolism Reverse Transcription/*physiology Two-Hybrid System Techniques Virion/metabolism Virus Replication/physiology, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Like all retroviruses, human immunodeficiency virus type 1 (HIV-1) undergoes reverse transcription during its replication cycle. The cellular cofactors potentially involved in this process still remain to be identified. We show here that A-kinase anchoring protein 149 (AKAP149) interacts with HIV-1 reverse transcriptase (RT) in both the yeast two-hybrid system and human cells. The AKAP149 binding site has been mapped to the RNase H domain of HIV-1 RT. AKAP149 silencing by RNA interference in HIV-1-infected cells inhibited viral replication at the reverse transcription step. We selected single-point mutants of RT defective for AKAP149 binding and demonstrated that mutant G462R, despite retaining significant intrinsic RT activity in vitro, failed to carry out HIV-1 reverse transcription correctly in infected cells. This suggests that the interaction between RT and AKAP149 in infected cells may play an important role in HIV-1 reverse transcription.
Lemay J, Maidou-Peindara P, Bader T, Ennifar E, Rain J C, Benarous R, Liu L X
HuR interacts with human immunodeficiency virus type 1 reverse transcriptase, and modulates reverse transcription in infected cells Article de journal
Dans: Retrovirology, vol. 5, p. 47, 2008, ISBN: 18544151, (1742-4690 (Electronic) Journal Article Research Support, Non-U.S. Gov't).
Résumé | Liens | BibTeX | Étiquettes: DUMAS Antigens, ENNIFAR, Small Interfering/genetics/metabolism RNA, Surface/*metabolism Binding Sites Cell Line Chromatin Immunoprecipitation Fluoroimmunoassay Gene Silencing HIV Reverse Transcriptase/*metabolism HIV-1/*physiology Humans Protein Interaction Domains and Motifs *Protein Interaction Mapping RNA, Unité ARN, Viral/metabolism RNA-Binding Proteins/*metabolism Two-Hybrid System Techniques *Virus Replication
@article{,
title = {HuR interacts with human immunodeficiency virus type 1 reverse transcriptase, and modulates reverse transcription in infected cells},
author = {J Lemay and P Maidou-Peindara and T Bader and E Ennifar and J C Rain and R Benarous and L X Liu},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18544151
http://www.retrovirology.com/content/5/1/47},
isbn = {18544151},
year = {2008},
date = {2008-01-01},
journal = {Retrovirology},
volume = {5},
pages = {47},
abstract = {Reverse transcription of the genetic material of human immunodeficiency virus type 1 (HIV-1) is a critical step in the replication cycle of this virus. This process, catalyzed by reverse transcriptase (RT), is well characterized at the biochemical level. However, in infected cells, reverse transcription occurs in a multiprotein complex - the reverse transcription complex (RTC) - consisting of viral genomic RNA associated with viral proteins (including RT) and, presumably, as yet uncharacterized cellular proteins. Very little is known about the cellular proteins interacting with the RTC, and with reverse transcriptase in particular. We report here that HIV-1 reverse transcription is affected by the levels of a nucleocytoplasmic shuttling protein - the RNA-binding protein HuR. A direct protein-protein interaction between RT and HuR was observed in a yeast two-hybrid screen and confirmed in vitro by homogenous time-resolved fluorescence (HTRF). We mapped the domain interacting with HuR to the RNAse H domain of RT, and the binding domain for RT to the C-terminus of HuR, partially overlapping the third RRM RNA-binding domain of HuR. HuR silencing with specific siRNAs greatly impaired early and late steps of reverse transcription, significantly inhibiting HIV-1 infection. Moreover, by mutagenesis and immunoprecipitation studies, we could not detect the binding of HuR to the viral RNA. These results suggest that HuR may be involved in and may modulate the reverse transcription reaction of HIV-1, by an as yet unknown mechanism involving a protein-protein interaction with HIV-1 RT.},
note = {1742-4690 (Electronic)
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {DUMAS Antigens, ENNIFAR, Small Interfering/genetics/metabolism RNA, Surface/*metabolism Binding Sites Cell Line Chromatin Immunoprecipitation Fluoroimmunoassay Gene Silencing HIV Reverse Transcriptase/*metabolism HIV-1/*physiology Humans Protein Interaction Domains and Motifs *Protein Interaction Mapping RNA, Unité ARN, Viral/metabolism RNA-Binding Proteins/*metabolism Two-Hybrid System Techniques *Virus Replication},
pubstate = {published},
tppubtype = {article}
}
Reverse transcription of the genetic material of human immunodeficiency virus type 1 (HIV-1) is a critical step in the replication cycle of this virus. This process, catalyzed by reverse transcriptase (RT), is well characterized at the biochemical level. However, in infected cells, reverse transcription occurs in a multiprotein complex - the reverse transcription complex (RTC) - consisting of viral genomic RNA associated with viral proteins (including RT) and, presumably, as yet uncharacterized cellular proteins. Very little is known about the cellular proteins interacting with the RTC, and with reverse transcriptase in particular. We report here that HIV-1 reverse transcription is affected by the levels of a nucleocytoplasmic shuttling protein - the RNA-binding protein HuR. A direct protein-protein interaction between RT and HuR was observed in a yeast two-hybrid screen and confirmed in vitro by homogenous time-resolved fluorescence (HTRF). We mapped the domain interacting with HuR to the RNAse H domain of RT, and the binding domain for RT to the C-terminus of HuR, partially overlapping the third RRM RNA-binding domain of HuR. HuR silencing with specific siRNAs greatly impaired early and late steps of reverse transcription, significantly inhibiting HIV-1 infection. Moreover, by mutagenesis and immunoprecipitation studies, we could not detect the binding of HuR to the viral RNA. These results suggest that HuR may be involved in and may modulate the reverse transcription reaction of HIV-1, by an as yet unknown mechanism involving a protein-protein interaction with HIV-1 RT.
Freisz S, Lang K, Micura R, Dumas P, Ennifar E
Binding of aminoglycoside antibiotics to the duplex form of the HIV-1 genomic RNA dimerization initiation site Article de journal
Dans: Angew Chem Int Ed Engl, vol. 47, no. 22, p. 4110-4113, 2008, ISBN: 18435520, (1521-3773 (Electronic) Journal Article Research Support, Non-U.S. Gov't).
Liens | BibTeX | Étiquettes: DUMAS Aminoglycosides/*chemistry/pharmacology Anti-Bacterial Agents/*chemistry/pharmacology Base Sequence Dimerization Genome, ENNIFAR, Unité ARN, Viral HIV-1/*drug effects Humans *Nucleic Acid Conformation RNA, Viral/*chemistry Virus Replication/*drug effects
@article{,
title = {Binding of aminoglycoside antibiotics to the duplex form of the HIV-1 genomic RNA dimerization initiation site},
author = {S Freisz and K Lang and R Micura and P Dumas and E Ennifar},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18435520},
isbn = {18435520},
year = {2008},
date = {2008-01-01},
journal = {Angew Chem Int Ed Engl},
volume = {47},
number = {22},
pages = {4110-4113},
note = {1521-3773 (Electronic)
Journal Article
Research Support, Non-U.S. Gov't},
keywords = {DUMAS Aminoglycosides/*chemistry/pharmacology Anti-Bacterial Agents/*chemistry/pharmacology Base Sequence Dimerization Genome, ENNIFAR, Unité ARN, Viral HIV-1/*drug effects Humans *Nucleic Acid Conformation RNA, Viral/*chemistry Virus Replication/*drug effects},
pubstate = {published},
tppubtype = {article}
}
2007
Bodlenner A, Alix A, Weibel J M, Pale P, Ennifar E, Paillart J C, Walter P, Marquet R, Dumas P
Synthesis of a neamine dimer targeting the dimerization initiation site of HIV-1 RNA Article de journal
Dans: Org Lett, vol. 9, no. 22, p. 4415-4418, 2007, ISBN: 17915882.
Résumé | Liens | BibTeX | Étiquettes: ENNIFAR, MARQUET, PAILLART, Unité ARN
@article{,
title = {Synthesis of a neamine dimer targeting the dimerization initiation site of HIV-1 RNA},
author = {A Bodlenner and A Alix and J M Weibel and P Pale and E Ennifar and J C Paillart and P Walter and R Marquet and P Dumas},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17915882},
isbn = {17915882},
year = {2007},
date = {2007-01-01},
journal = {Org Lett},
volume = {9},
number = {22},
pages = {4415-4418},
abstract = {A neamine dimer designed to bind to a specific sequence of HIV-1 RNA has been synthesized. Starting from neomycin B (1), a five-step synthesis efficiently provided a key protected neamine monomer 6 (28%). From the latter, coupling reactions with activated diacids gave dimers. After deprotection, a neamine dimer was obtained as the hexachlorohydrate salt 15 with 13% overall yield over nine steps.},
keywords = {ENNIFAR, MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
A neamine dimer designed to bind to a specific sequence of HIV-1 RNA has been synthesized. Starting from neomycin B (1), a five-step synthesis efficiently provided a key protected neamine monomer 6 (28%). From the latter, coupling reactions with activated diacids gave dimers. After deprotection, a neamine dimer was obtained as the hexachlorohydrate salt 15 with 13% overall yield over nine steps.
Ennifar E, Paillart J C, Bernacchi S, Walter P, Pale P, Decout J L, Marquet R, Dumas P
A structure-based approach for targeting the HIV-1 genomic RNA dimerization initiation site Article de journal
Dans: Biochimie, vol. 89, no. 10, p. 1195-1203, 2007, ISBN: 17434658.
Résumé | Liens | BibTeX | Étiquettes: ENNIFAR, MARQUET, PAILLART, Unité ARN
@article{,
title = {A structure-based approach for targeting the HIV-1 genomic RNA dimerization initiation site},
author = {E Ennifar and J C Paillart and S Bernacchi and P Walter and P Pale and J L Decout and R Marquet and P Dumas},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17434658},
isbn = {17434658},
year = {2007},
date = {2007-01-01},
journal = {Biochimie},
volume = {89},
number = {10},
pages = {1195-1203},
abstract = {Dimerization of the genomic RNA is an important step of the HIV-1 replication cycle. The Dimerization Initiation Site (DIS) promotes dimerization of the viral genome by forming a loop-loop complex between two DIS hairpins. Crystal structures of the DIS loop-loop complex revealed an unexpected and strong similitude with the bacterial 16S ribosomal aminoacyl-tRNA site (A site), which is the target of aminoglycoside antibiotics. As a consequence of these structural and sequence similarities, the HIV-1 DIS also binds some aminoglycosides, not only in vitro, but also ex vivo, in lymphoid cells and in viral particles. Crystal structures of the DIS loop-loop in complex with several aminoglycoside antibiotics provide a detailed-view of the DIS/drug interaction and reveal some hints about possible modifications to increase the drug affinity and/or specificity.},
keywords = {ENNIFAR, MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Dimerization of the genomic RNA is an important step of the HIV-1 replication cycle. The Dimerization Initiation Site (DIS) promotes dimerization of the viral genome by forming a loop-loop complex between two DIS hairpins. Crystal structures of the DIS loop-loop complex revealed an unexpected and strong similitude with the bacterial 16S ribosomal aminoacyl-tRNA site (A site), which is the target of aminoglycoside antibiotics. As a consequence of these structural and sequence similarities, the HIV-1 DIS also binds some aminoglycosides, not only in vitro, but also ex vivo, in lymphoid cells and in viral particles. Crystal structures of the DIS loop-loop in complex with several aminoglycoside antibiotics provide a detailed-view of the DIS/drug interaction and reveal some hints about possible modifications to increase the drug affinity and/or specificity.
Ali M B, Chaminade F, Kanevsky I, Ennifar E, Josset L, Ficheux D, Darlix J L, Fosse P
Structural requirements for nucleocapsid protein-mediated dimerization of avian leukosis virus RNA Article de journal
Dans: J Mol Biol, vol. 372, no. 4, p. 1082-96, 2007, ISBN: 17706668.
Résumé | Liens | BibTeX | Étiquettes: DUMAS, ENNIFAR, Unité ARN
@article{,
title = {Structural requirements for nucleocapsid protein-mediated dimerization of avian leukosis virus RNA},
author = {M B Ali and F Chaminade and I Kanevsky and E Ennifar and L Josset and D Ficheux and J L Darlix and P Fosse},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17706668},
isbn = {17706668},
year = {2007},
date = {2007-01-01},
journal = {J Mol Biol},
volume = {372},
number = {4},
pages = {1082-96},
abstract = {The avian leukosis virus (ALV) belongs to the alpha group of retroviruses that are widespread in nature. The 5'-untranslated region of ALV genome contains the L3 element that is important for virus infectivity and the formation of an unstable RNA dimer in vitro. The L3 sequence is predicted to fold into a long stem-loop structure with two internal loops and an apical one. Phylogenetic analysis predicts that the L3 stem-loop is conserved in alpharetroviruses. Furthermore, a significant selection mechanism maintains a palindrome in the apical loop. The nucleocapsid protein of the alpharetroviruses (NCp12) is required for RNA dimer formation and replication in vivo. It is not known whether L3 can be an NCp12-mediated RNA dimerization site able to bind NCp12 with high affinity. Here, we report that NCp12 chaperones formation of a stable ALV RNA dimer through L3. To investigate the NCp12-mediated L3 dimerization reaction, we performed site-directed mutagenesis, gel retardation and heterodimerization assays and analysis of thermostability of dimeric RNAs. We show that the affinity of NCp12 for L3 is lower than its affinity for the microPsi RNA packaging signal. Results show that conservation of a long stem-loop structure and a loop-loop interaction are not required for NCp12-mediated L3 dimerization. We show that the L3 apical stem-loop is sufficient to form an extended duplex and the whole stem-loop L3 cannot be converted by NCp12 into a duplex extending throughout L3. Three-dimensional modelling of the stable L3 dimer supports the notion that the extended duplex may represent the minimal dimer linkage structure found in the genomic RNA.},
keywords = {DUMAS, ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The avian leukosis virus (ALV) belongs to the alpha group of retroviruses that are widespread in nature. The 5'-untranslated region of ALV genome contains the L3 element that is important for virus infectivity and the formation of an unstable RNA dimer in vitro. The L3 sequence is predicted to fold into a long stem-loop structure with two internal loops and an apical one. Phylogenetic analysis predicts that the L3 stem-loop is conserved in alpharetroviruses. Furthermore, a significant selection mechanism maintains a palindrome in the apical loop. The nucleocapsid protein of the alpharetroviruses (NCp12) is required for RNA dimer formation and replication in vivo. It is not known whether L3 can be an NCp12-mediated RNA dimerization site able to bind NCp12 with high affinity. Here, we report that NCp12 chaperones formation of a stable ALV RNA dimer through L3. To investigate the NCp12-mediated L3 dimerization reaction, we performed site-directed mutagenesis, gel retardation and heterodimerization assays and analysis of thermostability of dimeric RNAs. We show that the affinity of NCp12 for L3 is lower than its affinity for the microPsi RNA packaging signal. Results show that conservation of a long stem-loop structure and a loop-loop interaction are not required for NCp12-mediated L3 dimerization. We show that the L3 apical stem-loop is sufficient to form an extended duplex and the whole stem-loop L3 cannot be converted by NCp12 into a duplex extending throughout L3. Three-dimensional modelling of the stable L3 dimer supports the notion that the extended duplex may represent the minimal dimer linkage structure found in the genomic RNA.
Bernacchi S, Freisz S, Maechling C, Spiess B, Marquet R, Dumas P, Ennifar E
Aminoglycoside binding to the HIV-1 RNA dimerization initiation site: thermodynamics and effect on the kissing-loop to duplex conversion Article de journal
Dans: Nucleic Acids Res, vol. 35, no. 21, p. 7128-39, 2007, ISBN: 17942426, (1362-4962 (Electronic)).
Résumé | Liens | BibTeX | Étiquettes: DUMAS, ENNIFAR, MARQUET, Unité ARN
@article{,
title = {Aminoglycoside binding to the HIV-1 RNA dimerization initiation site: thermodynamics and effect on the kissing-loop to duplex conversion},
author = {S Bernacchi and S Freisz and C Maechling and B Spiess and R Marquet and P Dumas and E Ennifar},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17942426},
isbn = {17942426},
year = {2007},
date = {2007-01-01},
journal = {Nucleic Acids Res},
volume = {35},
number = {21},
pages = {7128-39},
abstract = {Owing to a striking, and most likely fortuitous, structural and sequence similarity with the bacterial 16 S ribosomal A site, the RNA kissing-loop complex formed by the HIV-1 genomic RNA dimerization initiation site (DIS) specifically binds 4,5-disubstituted 2-deoxystreptamine (2-DOS) aminoglycoside antibiotics. We used chemical probing, molecular modeling, isothermal titration calorimetry (ITC) and UV melting to investigate aminoglycoside binding to the DIS loop-loop complex. We showed that apramycin, an aminoglycoside containing a bicyclic moiety, also binds the DIS, but in a different way than 4,5-disubstituted 2-DOS aminoglycosides. The determination of thermodynamic parameters for various aminoglycosides revealed the role of the different rings in the drug-RNA interaction. Surprisingly, we found that the affinity of lividomycin and neomycin for the DIS (K(d) approximately 30 nM) is significantly higher than that obtained in the same experimental conditions for their natural target, the bacterial A site (K(d) approximately 1.6 microM). In good agreement with their respective affinity, aminoglycoside increase the melting temperature of the loop-loop interaction and also block the conversion from kissing-loop complex to extended duplex. Taken together, our data might be useful for selecting new molecules with improved specificity and affinity toward the HIV-1 DIS RNA.},
note = {1362-4962 (Electronic)},
keywords = {DUMAS, ENNIFAR, MARQUET, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Owing to a striking, and most likely fortuitous, structural and sequence similarity with the bacterial 16 S ribosomal A site, the RNA kissing-loop complex formed by the HIV-1 genomic RNA dimerization initiation site (DIS) specifically binds 4,5-disubstituted 2-deoxystreptamine (2-DOS) aminoglycoside antibiotics. We used chemical probing, molecular modeling, isothermal titration calorimetry (ITC) and UV melting to investigate aminoglycoside binding to the DIS loop-loop complex. We showed that apramycin, an aminoglycoside containing a bicyclic moiety, also binds the DIS, but in a different way than 4,5-disubstituted 2-DOS aminoglycosides. The determination of thermodynamic parameters for various aminoglycosides revealed the role of the different rings in the drug-RNA interaction. Surprisingly, we found that the affinity of lividomycin and neomycin for the DIS (K(d) approximately 30 nM) is significantly higher than that obtained in the same experimental conditions for their natural target, the bacterial A site (K(d) approximately 1.6 microM). In good agreement with their respective affinity, aminoglycoside increase the melting temperature of the loop-loop interaction and also block the conversion from kissing-loop complex to extended duplex. Taken together, our data might be useful for selecting new molecules with improved specificity and affinity toward the HIV-1 DIS RNA.
Ennifar E, Bernacchi S, Wolff P, Dumas P
Influence of C-5 halogenation of uridines on hairpin versus duplex RNA folding. Article de journal
Dans: RNA, vol. 13, no. 9, p. 1445-52, 2007, ISBN: 17630326.
Résumé | Liens | BibTeX | Étiquettes: ENNIFAR, MARQUET, RNA HIV fluorescence halogen crystal, Unité ARN
@article{,
title = {Influence of C-5 halogenation of uridines on hairpin versus duplex RNA folding.},
author = {E Ennifar and S Bernacchi and P Wolff and P Dumas},
url = {http://www.ncbi.nlm.nih.gov/pubmed/17630326},
isbn = {17630326},
year = {2007},
date = {2007-01-01},
journal = {RNA},
volume = {13},
number = {9},
pages = {1445-52},
abstract = {Halogenation of bases is a widespread method used for solving crystal structures of nucleic acids. However, this modification may have important consequences on RNA folding and thus on the success of crystallization. We have used a combination of UV thermal melting, steady-state fluorescence, X-ray crystallography, and gel electrophoresis techniques to study the influence of uridine halogenation (bromination or iodination) on the RNA folding. The HIV-1 Dimerization Initiation Site is an RNA hairpin that can adopt an alternative duplex conformation and was used as a model. We have shown that, unexpectedly, the RNA hairpin/duplex ratio is strongly dependent not only on the presence but also on the position of halogenation.},
keywords = {ENNIFAR, MARQUET, RNA HIV fluorescence halogen crystal, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Halogenation of bases is a widespread method used for solving crystal structures of nucleic acids. However, this modification may have important consequences on RNA folding and thus on the success of crystallization. We have used a combination of UV thermal melting, steady-state fluorescence, X-ray crystallography, and gel electrophoresis techniques to study the influence of uridine halogenation (bromination or iodination) on the RNA folding. The HIV-1 Dimerization Initiation Site is an RNA hairpin that can adopt an alternative duplex conformation and was used as a model. We have shown that, unexpectedly, the RNA hairpin/duplex ratio is strongly dependent not only on the presence but also on the position of halogenation.
Olieric V, Ennifar E, Meents A, Fleurant M, Besnard C, Pattison P, Schiltz M, Schulze-Briese C, Dumas P
Using X-ray absorption spectra to monitor specific radiation damage to anomalously scattering atoms in macromolecular crystallography Article de journal
Dans: Acta Crystallogr D Biol Crystallogr, vol. 63, no. Pt 7, p. 759-68, 2007, ISBN: 17582167.
Résumé | Liens | BibTeX | Étiquettes: DUMAS, ENNIFAR, Unité ARN
@article{,
title = {Using X-ray absorption spectra to monitor specific radiation damage to anomalously scattering atoms in macromolecular crystallography},
author = {V Olieric and E Ennifar and A Meents and M Fleurant and C Besnard and P Pattison and M Schiltz and C Schulze-Briese and P Dumas},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17582167},
isbn = {17582167},
year = {2007},
date = {2007-01-01},
journal = {Acta Crystallogr D Biol Crystallogr},
volume = {63},
number = {Pt 7},
pages = {759-68},
abstract = {Radiation damage in macromolecular crystals is not suppressed even at 90 K. This is particularly true for covalent bonds involving an anomalous scatterer (such as bromine) at the 'peak wavelength'. It is shown that a series of absorption spectra recorded on a brominated RNA faithfully monitor the extent of cleavage. The continuous spectral changes during irradiation preserve an 'isosbestic point', each spectrum being a linear combination of 'zero' and 'infinite' dose spectra. This easily yields a good estimate of the partial occupancy of bromine at any intermediate dose. The considerable effect on the near-edge features in the spectra of the crystal orientation versus the beam polarization has also been examined and found to be in good agreement with a previous study. Any significant influence of the (C-Br bond/beam polarization) angle on the cleavage kinetics of bromine was also searched for, but was not detected. These results will be useful for standard SAD/MAD experiments and for the emerging 'radiation-damage-induced phasing' method exploiting both the anomalous signal of an anomalous scatterer and the 'isomorphous' signal resulting from its cleavage.},
keywords = {DUMAS, ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Radiation damage in macromolecular crystals is not suppressed even at 90 K. This is particularly true for covalent bonds involving an anomalous scatterer (such as bromine) at the 'peak wavelength'. It is shown that a series of absorption spectra recorded on a brominated RNA faithfully monitor the extent of cleavage. The continuous spectral changes during irradiation preserve an 'isosbestic point', each spectrum being a linear combination of 'zero' and 'infinite' dose spectra. This easily yields a good estimate of the partial occupancy of bromine at any intermediate dose. The considerable effect on the near-edge features in the spectra of the crystal orientation versus the beam polarization has also been examined and found to be in good agreement with a previous study. Any significant influence of the (C-Br bond/beam polarization) angle on the cleavage kinetics of bromine was also searched for, but was not detected. These results will be useful for standard SAD/MAD experiments and for the emerging 'radiation-damage-induced phasing' method exploiting both the anomalous signal of an anomalous scatterer and the 'isomorphous' signal resulting from its cleavage.
Reblova K, Fadrna E, Sarzynska J, Kulinski T, Kulhanek P, Ennifar E, Koca J, Sponer J
Conformations of Flanking Bases in HIV-1 RNA DIS Kissing Complexes Studied by Molecular Dynamics Article de journal
Dans: Biophys J, vol. 93, no. 11, p. 3932-3949, 2007, ISBN: 17704156, (0006-3495 (Print) Journal article).
Résumé | Liens | BibTeX | Étiquettes: DUMAS, ENNIFAR, Unité ARN
@article{,
title = {Conformations of Flanking Bases in HIV-1 RNA DIS Kissing Complexes Studied by Molecular Dynamics},
author = {K Reblova and E Fadrna and J Sarzynska and T Kulinski and P Kulhanek and E Ennifar and J Koca and J Sponer},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17704156},
isbn = {17704156},
year = {2007},
date = {2007-01-01},
journal = {Biophys J},
volume = {93},
number = {11},
pages = {3932-3949},
abstract = {Explicit solvent molecular dynamics simulations (in total almost 800 ns including locally enhanced sampling runs) were applied with different ion conditions and with two force fields (AMBER and CHARMM) to characterize typical geometries adopted by the flanking bases in the RNA kissing-loop complexes. We focus on flanking base positions in multiple x-ray and NMR structures of HIV-1 DIS kissing complexes and kissing complex from the large ribosomal subunit of Haloarcula marismortui. An initial x-ray open conformation of bulged-out bases in HIV-1 DIS complexes, affected by crystal packing, tends to convert to a closed conformation formed by consecutive stretch of four stacked purine bases. This is in agreement with those recent crystals where the packing is essentially avoided. We also observed variants of the closed conformation with three stacked bases, while nonnegligible populations of stacked geometries with bulged-in bases were detected, too. The simulation results reconcile differences in positions of the flanking bases observed in x-ray and NMR studies. Our results suggest that bulged-out geometries are somewhat more preferred, which is in accord with recent experiments showing that they may mediate tertiary contacts in biomolecular assemblies or allow binding of aminoglycoside antibiotics.},
note = {0006-3495 (Print)
Journal article},
keywords = {DUMAS, ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Explicit solvent molecular dynamics simulations (in total almost 800 ns including locally enhanced sampling runs) were applied with different ion conditions and with two force fields (AMBER and CHARMM) to characterize typical geometries adopted by the flanking bases in the RNA kissing-loop complexes. We focus on flanking base positions in multiple x-ray and NMR structures of HIV-1 DIS kissing complexes and kissing complex from the large ribosomal subunit of Haloarcula marismortui. An initial x-ray open conformation of bulged-out bases in HIV-1 DIS complexes, affected by crystal packing, tends to convert to a closed conformation formed by consecutive stretch of four stacked purine bases. This is in agreement with those recent crystals where the packing is essentially avoided. We also observed variants of the closed conformation with three stacked bases, while nonnegligible populations of stacked geometries with bulged-in bases were detected, too. The simulation results reconcile differences in positions of the flanking bases observed in x-ray and NMR studies. Our results suggest that bulged-out geometries are somewhat more preferred, which is in accord with recent experiments showing that they may mediate tertiary contacts in biomolecular assemblies or allow binding of aminoglycoside antibiotics.
2006
Ennifar E, Dumas P
Polymorphism of bulged-out residues in HIV-1 RNA DIS kissing complex and structure comparison with solution studies Article de journal
Dans: J Mol Biol, vol. 356, no. 3, p. 771-782, 2006, ISBN: 16403527, (0022-2836 (Print) Journal Article).
Résumé | Liens | BibTeX | Étiquettes: DUMAS, ENNIFAR, Unité ARN
@article{,
title = {Polymorphism of bulged-out residues in HIV-1 RNA DIS kissing complex and structure comparison with solution studies},
author = {E Ennifar and P Dumas},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16403527},
isbn = {16403527},
year = {2006},
date = {2006-01-01},
journal = {J Mol Biol},
volume = {356},
number = {3},
pages = {771-782},
abstract = {All retroviruses encapsidate their genome as a dimer of homologous single-stranded RNAs. The dimerization initiation site (DIS) of human immunodeficiency virus type 1 (HIV-1) is located in the 5'-untranslated region of the viral genome and consists of a hairpin with a 6 nt self-complementary loop sequence. Genomic RNA dimerization, a crucial step for virion infectivity, is promoted by the formation of a loop-loop complex (or kissing complex) between two DIS hairpins. Crystal structures for the subtypes A, B and F of the HIV-1 DIS kissing complex have now been solved at 2.3 A, 1.9 A and 1.6 A, respectively. They revealed a polymorphism of bulged-out residues showing clearly that their conformation is not a mere consequence of crystal packing. They also provide more insights into ion binding, hydration, and RNA conformation and flexibility. In particular, we observed the binding of spermine to the loop-loop helix, which displaced a magnesium cation important for subtype A DIS dimerization. The excellent agreement between X-ray structures and the results of chemical probing and interference data on larger viral RNA fragments shows that the crystal structures are relevant for the DIS kissing complex present in solution and in viral particles. Accordingly, these structures will be helpful for designing new drugs derived from aminoglycoside antibiotics and targeted against the RNA dimerization step of the viral life-cycle.},
note = {0022-2836 (Print)
Journal Article},
keywords = {DUMAS, ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
All retroviruses encapsidate their genome as a dimer of homologous single-stranded RNAs. The dimerization initiation site (DIS) of human immunodeficiency virus type 1 (HIV-1) is located in the 5'-untranslated region of the viral genome and consists of a hairpin with a 6 nt self-complementary loop sequence. Genomic RNA dimerization, a crucial step for virion infectivity, is promoted by the formation of a loop-loop complex (or kissing complex) between two DIS hairpins. Crystal structures for the subtypes A, B and F of the HIV-1 DIS kissing complex have now been solved at 2.3 A, 1.9 A and 1.6 A, respectively. They revealed a polymorphism of bulged-out residues showing clearly that their conformation is not a mere consequence of crystal packing. They also provide more insights into ion binding, hydration, and RNA conformation and flexibility. In particular, we observed the binding of spermine to the loop-loop helix, which displaced a magnesium cation important for subtype A DIS dimerization. The excellent agreement between X-ray structures and the results of chemical probing and interference data on larger viral RNA fragments shows that the crystal structures are relevant for the DIS kissing complex present in solution and in viral particles. Accordingly, these structures will be helpful for designing new drugs derived from aminoglycoside antibiotics and targeted against the RNA dimerization step of the viral life-cycle.
Ennifar E, Paillart J C, Bodlenner A, Walter P, Weibel J M, Aubertin A M, Pale P, Dumas P, Marquet R
Targeting the dimerization initiation site of HIV-1 RNA with aminoglycosides: from crystal to cell Article de journal
Dans: Nucleic Acids Research, vol. 34, no. 8, p. 2328-39, 2006.
Résumé | Liens | BibTeX | Étiquettes: Aminoglycosides/*chemistry Anti-HIV Agents/*chemistry Binding Sites Cell Line Crystallography, ENNIFAR, MARQUET, Molecular RNA, Non-U.S. Gov't Virion/chemistry, PAILLART, Unité ARN, Viral/*chemistry Research Support, X-Ray Dimerization Drug Delivery Systems HIV-1/*genetics Humans Models
@article{Ennifar2006,
title = {Targeting the dimerization initiation site of HIV-1 RNA with aminoglycosides: from crystal to cell},
author = {E Ennifar and J C Paillart and A Bodlenner and P Walter and J M Weibel and A M Aubertin and P Pale and P Dumas and R Marquet},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16679451},
doi = {10.1093/nar/gkl317 },
year = {2006},
date = {2006-01-01},
journal = {Nucleic Acids Research},
volume = {34},
number = {8},
pages = {2328-39},
abstract = {The kissing-loop complex that initiates dimerization of genomic RNA is crucial for Human Immunodeficiency Virus Type 1 (HIV-1) replication. We showed that owing to its strong similitude with the bacterial ribosomal A site it can be targeted by aminoglycosides. Here, we present its crystal structure in complex with neamine, ribostamycin, neomycin and lividomycin. These structures explain the specificity for 4,5-disubstituted 2-deoxystreptamine (DOS) derivatives and for subtype A and subtype F kissing-loop complexes, and provide a strong basis for rational drug design. As a consequence of the different topologies of the kissing-loop complex and the A site, these aminoglycosides establish more contacts with HIV-1 RNA than with 16S RNA. Together with biochemical experiments, they showed that while rings I, II and III confer binding specificity, rings IV and V are important for affinity. Binding of neomycin, paromomycin and lividomycin strongly stabilized the kissing-loop complex by bridging the two HIV-1 RNA molecules. Furthermore, in situ footprinting showed that the dimerization initiation site (DIS) of HIV-1 genomic RNA could be targeted by these aminoglycosides in infected cells and virions, demonstrating its accessibility.},
keywords = {Aminoglycosides/*chemistry Anti-HIV Agents/*chemistry Binding Sites Cell Line Crystallography, ENNIFAR, MARQUET, Molecular RNA, Non-U.S. Gov't Virion/chemistry, PAILLART, Unité ARN, Viral/*chemistry Research Support, X-Ray Dimerization Drug Delivery Systems HIV-1/*genetics Humans Models},
pubstate = {published},
tppubtype = {article}
}
The kissing-loop complex that initiates dimerization of genomic RNA is crucial for Human Immunodeficiency Virus Type 1 (HIV-1) replication. We showed that owing to its strong similitude with the bacterial ribosomal A site it can be targeted by aminoglycosides. Here, we present its crystal structure in complex with neamine, ribostamycin, neomycin and lividomycin. These structures explain the specificity for 4,5-disubstituted 2-deoxystreptamine (DOS) derivatives and for subtype A and subtype F kissing-loop complexes, and provide a strong basis for rational drug design. As a consequence of the different topologies of the kissing-loop complex and the A site, these aminoglycosides establish more contacts with HIV-1 RNA than with 16S RNA. Together with biochemical experiments, they showed that while rings I, II and III confer binding specificity, rings IV and V are important for affinity. Binding of neomycin, paromomycin and lividomycin strongly stabilized the kissing-loop complex by bridging the two HIV-1 RNA molecules. Furthermore, in situ footprinting showed that the dimerization initiation site (DIS) of HIV-1 genomic RNA could be targeted by these aminoglycosides in infected cells and virions, demonstrating its accessibility.
2005
Ennifar E, Basquin J, Birkenbihl R, Suck D
Purification, crystallization and preliminary X-ray diffraction studies of the archaeal virus resolvase SIRV2 Article de journal
Dans: Acta Crystallogr F Struct Biol Commun, vol. 61, no. Pt 5, p. 507-509, 2005, ISBN: 16511081, (1744-3091 (Electronic) Journal Article).
Résumé | Liens | BibTeX | Étiquettes: DUMAS, ENNIFAR, Unité ARN
@article{,
title = {Purification, crystallization and preliminary X-ray diffraction studies of the archaeal virus resolvase SIRV2},
author = {E Ennifar and J Basquin and R Birkenbihl and D Suck},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16511081},
isbn = {16511081},
year = {2005},
date = {2005-01-01},
journal = {Acta Crystallogr F Struct Biol Commun},
volume = {61},
number = {Pt 5},
pages = {507-509},
abstract = {The Holliday junction (or four-way junction) is the universal DNA intermediate whose interaction with resolving proteins is one of the major events in the recombinational process. These proteins, called DNA junction-resolving enzymes or resolvases, bind to the junction and catalyse DNA cleavage, promoting the release of two DNA duplexes. SIRV2 Hjc, a viral resolvase infecting a thermophylic archaeon, has been cloned, expressed and purified. Crystals have been obtained in space group C2, with unit-cell parameters a = 147.8},
note = {1744-3091 (Electronic)
Journal Article},
keywords = {DUMAS, ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The Holliday junction (or four-way junction) is the universal DNA intermediate whose interaction with resolving proteins is one of the major events in the recombinational process. These proteins, called DNA junction-resolving enzymes or resolvases, bind to the junction and catalyse DNA cleavage, promoting the release of two DNA duplexes. SIRV2 Hjc, a viral resolvase infecting a thermophylic archaeon, has been cloned, expressed and purified. Crystals have been obtained in space group C2, with unit-cell parameters a = 147.8
Bernacchi S, Ennifar E, Toth K, Walter P, Langowski J, Dumas P
Mechanism of hairpin-duplex conversion for the HIV-1 dimerization initiation site Article de journal
Dans: J Biol Chem, vol. 280, no. 48, p. 40112-40121, 2005, ISBN: 16169845, (0021-9258 (Print) Journal Article).
Résumé | Liens | BibTeX | Étiquettes: Base Sequence Binding Sites Crystallography, Drug Electrophoresis Genome, ENNIFAR, Fluorescence Spectrophotometry Temperature Thermodynamics Time Factors Ultraviolet Rays Virus Replication, MARQUET, Non-U.S. Gov't Spectrometry, PAILLART, Unité ARN, Viral HIV-1/*chemistry Kinetics Molecular Sequence Data *Nucleic Acid Conformation Protein Binding RNA/chemistry RNA, Viral/*chemistry Research Support, X-Ray Dimerization Dose-Response Relationship
@article{,
title = {Mechanism of hairpin-duplex conversion for the HIV-1 dimerization initiation site},
author = {S Bernacchi and E Ennifar and K Toth and P Walter and J Langowski and P Dumas},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16169845},
isbn = {16169845},
year = {2005},
date = {2005-01-01},
journal = {J Biol Chem},
volume = {280},
number = {48},
pages = {40112-40121},
abstract = {We have used the dimerization initiation site of HIV-1 genomic RNA as a model to investigate hairpin-duplex interconversion with a combination of fluorescence, UV melting, gel electrophoresis, and x-ray crystallographic techniques. Fluorescence studies with molecular beacons and crystallization experiments with 23-nucleotide dimerization initiation site fragments showed that the ratio of hairpin to duplex formed after annealing in water essentially depends on RNA concentration and not on cooling kinetics. With natural sequences allowing to form the most stable duplex, and thus also the loop-loop complex (or "kissing complex"), concentrations as low as 3 mum in strands are necessary to obtain a majority of the hairpin form. With a mutated sequence preventing kissing complex formation, a majority of hairpins was even obtained at 80 mum in strands. However, this did not prevent an efficient conversion from hairpin to duplex in the presence of salts. Kinetic considerations are in favor of duplex formation from intermediates involving hairpins engaged in cruciform dimers rather than from free strands. The very first step of formation of such a cruciform intermediate could be trapped in a crystal structure. This mechanism might be significant for the dynamics of small RNAs beyond the strict field of HIV-1.},
note = {0021-9258 (Print)
Journal Article},
keywords = {Base Sequence Binding Sites Crystallography, Drug Electrophoresis Genome, ENNIFAR, Fluorescence Spectrophotometry Temperature Thermodynamics Time Factors Ultraviolet Rays Virus Replication, MARQUET, Non-U.S. Gov't Spectrometry, PAILLART, Unité ARN, Viral HIV-1/*chemistry Kinetics Molecular Sequence Data *Nucleic Acid Conformation Protein Binding RNA/chemistry RNA, Viral/*chemistry Research Support, X-Ray Dimerization Dose-Response Relationship},
pubstate = {published},
tppubtype = {article}
}
We have used the dimerization initiation site of HIV-1 genomic RNA as a model to investigate hairpin-duplex interconversion with a combination of fluorescence, UV melting, gel electrophoresis, and x-ray crystallographic techniques. Fluorescence studies with molecular beacons and crystallization experiments with 23-nucleotide dimerization initiation site fragments showed that the ratio of hairpin to duplex formed after annealing in water essentially depends on RNA concentration and not on cooling kinetics. With natural sequences allowing to form the most stable duplex, and thus also the loop-loop complex (or "kissing complex"), concentrations as low as 3 mum in strands are necessary to obtain a majority of the hairpin form. With a mutated sequence preventing kissing complex formation, a majority of hairpins was even obtained at 80 mum in strands. However, this did not prevent an efficient conversion from hairpin to duplex in the presence of salts. Kinetic considerations are in favor of duplex formation from intermediates involving hairpins engaged in cruciform dimers rather than from free strands. The very first step of formation of such a cruciform intermediate could be trapped in a crystal structure. This mechanism might be significant for the dynamics of small RNAs beyond the strict field of HIV-1.
Gaied N Ben, Glasser N, Ramalanjaona N, Beltz H, Wolff P, Marquet R, Burger A, Mely Y
8-vinyl-deoxyadenosine, an alternative fluorescent nucleoside analog to 2'-deoxyribosyl-2-aminopurine with improved properties Article de journal
Dans: Nucleic Acids Res, vol. 33, no. 3, p. 1031-1039, 2005, ISBN: 15718302, (1362-4962 Journal Article).
Résumé | Liens | BibTeX | Étiquettes: ENNIFAR, MARQUET, PAILLART, Unité ARN
@article{,
title = {8-vinyl-deoxyadenosine, an alternative fluorescent nucleoside analog to 2'-deoxyribosyl-2-aminopurine with improved properties},
author = {N Ben Gaied and N Glasser and N Ramalanjaona and H Beltz and P Wolff and R Marquet and A Burger and Y Mely},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15718302},
isbn = {15718302},
year = {2005},
date = {2005-01-01},
journal = {Nucleic Acids Res},
volume = {33},
number = {3},
pages = {1031-1039},
abstract = {We report here the synthesis and the spectroscopic characterization of 8-vinyl-deoxyadenosine (8vdA), a new fluorescent analog of deoxyadenosine. 8vdA was found to absorb and emit in the same wavelength range as 2'-deoxyribosyl-2-aminopurine (2AP), the most frequently used fluorescent nucleoside analog. Though the quantum yield of 8vdA is similar to that of 2AP, its molar absorption coefficient is about twice, enabling a more sensitive detection. Moreover, the fluorescence of 8vdA was found to be sensitive to temperature and solvent but not to pH (around neutrality) or coupling to phosphate groups. Though 8vdA is base sensitive and susceptible to depurination, the corresponding phosphoramidite was successfully prepared and incorporated in oligonucleotides of the type d(CGT TTT XNX TTT TGC) where N = 8vdA and X = A, T or C. The 8vdA-labeled oligonucleotides gave more stable duplexes than the corresponding 2AP-labeled sequences when X = A or T, indicating that 8vdA is less perturbing than 2AP and probably adopts an anti conformation to preserve the Watson-Crick H-bonding. In addition, the quantum yield of 8vdA is significantly higher than 2AP in all tested oligonucleotides in both their single strand and duplex states. The steady-state and time-resolved fluorescence parameters of 8vdA and 2AP were found to depend similarly on the nature of their flanking residues and on base pairing, suggesting that their photophysics are governed by similar mechanisms. Taken together, our data suggest that 8vdA is a non perturbing nucleoside analog that may be used with improved sensitivity for the same applications as 2AP.},
note = {1362-4962
Journal Article},
keywords = {ENNIFAR, MARQUET, PAILLART, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
We report here the synthesis and the spectroscopic characterization of 8-vinyl-deoxyadenosine (8vdA), a new fluorescent analog of deoxyadenosine. 8vdA was found to absorb and emit in the same wavelength range as 2'-deoxyribosyl-2-aminopurine (2AP), the most frequently used fluorescent nucleoside analog. Though the quantum yield of 8vdA is similar to that of 2AP, its molar absorption coefficient is about twice, enabling a more sensitive detection. Moreover, the fluorescence of 8vdA was found to be sensitive to temperature and solvent but not to pH (around neutrality) or coupling to phosphate groups. Though 8vdA is base sensitive and susceptible to depurination, the corresponding phosphoramidite was successfully prepared and incorporated in oligonucleotides of the type d(CGT TTT XNX TTT TGC) where N = 8vdA and X = A, T or C. The 8vdA-labeled oligonucleotides gave more stable duplexes than the corresponding 2AP-labeled sequences when X = A or T, indicating that 8vdA is less perturbing than 2AP and probably adopts an anti conformation to preserve the Watson-Crick H-bonding. In addition, the quantum yield of 8vdA is significantly higher than 2AP in all tested oligonucleotides in both their single strand and duplex states. The steady-state and time-resolved fluorescence parameters of 8vdA and 2AP were found to depend similarly on the nature of their flanking residues and on base pairing, suggesting that their photophysics are governed by similar mechanisms. Taken together, our data suggest that 8vdA is a non perturbing nucleoside analog that may be used with improved sensitivity for the same applications as 2AP.
2004
Schiltz M, Dumas P, Ennifar E, Flensburg C, Paciorek W, Vonrhein C, Bricogne G
Phasing in the presence of severe site-specific radiation damage through dose-dependent modelling of heavy atoms Article de journal
Dans: Acta Crystallogr D Biol Crystallogr, vol. 60, no. Pt 6, p. 1024-1031, 2004, ISBN: 15159561, (0907-4449 Journal Article).
Résumé | Liens | BibTeX | Étiquettes: DUMAS, ENNIFAR, Unité ARN
@article{,
title = {Phasing in the presence of severe site-specific radiation damage through dose-dependent modelling of heavy atoms},
author = {M Schiltz and P Dumas and E Ennifar and C Flensburg and W Paciorek and C Vonrhein and G Bricogne},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15159561},
isbn = {15159561},
year = {2004},
date = {2004-01-01},
journal = {Acta Crystallogr D Biol Crystallogr},
volume = {60},
number = {Pt 6},
pages = {1024-1031},
abstract = {The case of a brominated RNA crystal structure determination in which standard three-wavelength MAD phasing was unsuccessful because of fast X-ray-induced debromination was reinvestigated [Ennifar et al. (2002), Acta Cryst. D58, 1262-1268]. It was found that if the data are kept unmerged and if a dose-stamp is associated with each reflection measurement, dose-dependent occupancies can be refined for the Br atoms. Such a parametrization has been implemented in the macromolecular phasing program SHARP. Refining such dose-dependent occupancies on an unmerged data set gave a dramatic improvement, even for SAD phases from only the first wavelength (peak), and resulted in a good electron-density map after solvent flattening. The adverse effect of radiation damage has been turned into a beneficial one. The crucial difference is made by the use of unmerged data: phasing power is generated through the intensity differences of symmetry-related reflections recorded at different doses, i.e. corresponding to different states of the X-ray-induced debromination. This approach should prove useful in all situations of experimental phasing where site-specific radiation damage occurs unavoidably and undesirably and not only in cases in which radiation damage is purposely being created in order to demonstrate its potential usefulness.},
note = {0907-4449
Journal Article},
keywords = {DUMAS, ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The case of a brominated RNA crystal structure determination in which standard three-wavelength MAD phasing was unsuccessful because of fast X-ray-induced debromination was reinvestigated [Ennifar et al. (2002), Acta Cryst. D58, 1262-1268]. It was found that if the data are kept unmerged and if a dose-stamp is associated with each reflection measurement, dose-dependent occupancies can be refined for the Br atoms. Such a parametrization has been implemented in the macromolecular phasing program SHARP. Refining such dose-dependent occupancies on an unmerged data set gave a dramatic improvement, even for SAD phases from only the first wavelength (peak), and resulted in a good electron-density map after solvent flattening. The adverse effect of radiation damage has been turned into a beneficial one. The crucial difference is made by the use of unmerged data: phasing power is generated through the intensity differences of symmetry-related reflections recorded at different doses, i.e. corresponding to different states of the X-ray-induced debromination. This approach should prove useful in all situations of experimental phasing where site-specific radiation damage occurs unavoidably and undesirably and not only in cases in which radiation damage is purposely being created in order to demonstrate its potential usefulness.
Ehresmann C, Ehresmann B, Ennifar E, Dumas P, Garber M, Mathy N, Nikulin A, Portier C, Patel D, Serganov A
Molecular mimicry in translational regulation: the case of ribosomal protein S15. Article de journal
Dans: RNA Biol, vol. 1, no. 1, p. 66-73, 2004, ISBN: 17194931, (Epub 2004 May 5.).
Résumé | Liens | BibTeX | Étiquettes: DUMAS, ENNIFAR, Unité ARN
@article{,
title = {Molecular mimicry in translational regulation: the case of ribosomal protein S15.},
author = {C Ehresmann and B Ehresmann and E Ennifar and P Dumas and M Garber and N Mathy and A Nikulin and C Portier and D Patel and A Serganov},
url = {http://www.ncbi.nlm.nih.gov/pubmed/17194931},
isbn = {17194931},
year = {2004},
date = {2004-01-01},
journal = {RNA Biol},
volume = {1},
number = {1},
pages = {66-73},
abstract = {Ribosomal protein S15 is highly conserved among prokaryotes. It plays a pivotal role in the assembly of the central domain of the small ribosomal subunit and regulates its own expression by a feedback mechanism at the translational level. The protein recognizes two RNA targets (rRNA and mRNA) that share only partial similarity. Its interaction with 16S rRNA has been fully characterized, while mRNA interactions and regulatory mechanisms have been extensively studied in E. coli and in T. thermophilus. Recently, we have characterized which aminoacids are involved in E. coli mRNA recognition, using an in vivo assay allowing to identify S15 mutations affecting the S15-mRNA interactions without altering 30S subunit assembly. Here, we address the following questions: Are common determinants used by S15 to recognize its rRNA and mRNA targets? What is the extent of molecular mimicry? Is the regulatory mechanism conserved? Our results indicate that specific recognition of mRNA and rRNA relies on both mimicry and site differentiation. They also highlight the high plasticity of RNA to adapt to evolutionary constraints.},
note = {Epub 2004 May 5.},
keywords = {DUMAS, ENNIFAR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Ribosomal protein S15 is highly conserved among prokaryotes. It plays a pivotal role in the assembly of the central domain of the small ribosomal subunit and regulates its own expression by a feedback mechanism at the translational level. The protein recognizes two RNA targets (rRNA and mRNA) that share only partial similarity. Its interaction with 16S rRNA has been fully characterized, while mRNA interactions and regulatory mechanisms have been extensively studied in E. coli and in T. thermophilus. Recently, we have characterized which aminoacids are involved in E. coli mRNA recognition, using an in vivo assay allowing to identify S15 mutations affecting the S15-mRNA interactions without altering 30S subunit assembly. Here, we address the following questions: Are common determinants used by S15 to recognize its rRNA and mRNA targets? What is the extent of molecular mimicry? Is the regulatory mechanism conserved? Our results indicate that specific recognition of mRNA and rRNA relies on both mimicry and site differentiation. They also highlight the high plasticity of RNA to adapt to evolutionary constraints.
2003
Ennifar E, Walter P, Dumas P
A crystallographic study of the binding of 13 metal ions to two related RNA duplexes Article de journal
Dans: Nucleic Acids Res, vol. 31, no. 10, p. 2671-2682, 2003, ISBN: 12736317, (1362-4962 Journal Article).
Résumé | Liens | BibTeX | Étiquettes: Base Sequence Binding Sites/genetics Binding, Competitive Cations, Divalent/chemistry/metabolism Cobalt/chemistry/metabolism Comparative Study Crystallization Crystallography, ENNIFAR, Molecular Nucleic Acid Heteroduplexes/*chemistry/genetics/metabolism Oligoribonucleotides/chemistry/genetics/metabolism Platinum Compounds/chemistry/metabolism RNA/*chemistry/genetics/metabolism Ruthenium Compounds/chemistry/metabolism Support, Non-U.S. Gov't, Unité ARN, X-Ray Gold Compounds/chemistry/metabolism Magnesium/chemistry/metabolism Metals/*chemistry/metabolism Models
@article{,
title = {A crystallographic study of the binding of 13 metal ions to two related RNA duplexes},
author = {E Ennifar and P Walter and P Dumas},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12736317},
isbn = {12736317},
year = {2003},
date = {2003-01-01},
journal = {Nucleic Acids Res},
volume = {31},
number = {10},
pages = {2671-2682},
abstract = {Metal ions, and magnesium in particular, are known to be involved in RNA folding by stabilizing secondary and tertiary structures, and, as cofactors, in RNA enzymatic activity. We have conducted a systematic crystallographic analysis of cation binding to the duplex form of the HIV-1 RNA dimerization initiation site for the subtype-A and -B natural sequences. Eleven ions (K+, Pb2+, Mn2+, Ba2+, Ca2+, Cd2+, Sr2+, Zn2+, Co2+, Au3+ and Pt4+) and two hexammines [Co (NH3)6]3+ and [Ru (NH3)6]3+ were found to bind to the DIS duplex structure. Although the two sequences are very similar, strong differences were found in their cation binding properties. Divalent cations bind almost exclusively, as Mg2+, at 'Hoogsteen' sites of guanine residues, with a cation-dependent affinity for each site. Notably, a given cation can have very different affinities for a priori equivalent sites within the same molecule. Surprisingly, none of the two hexammines used were able to efficiently replace hexahydrated magnesium. Instead, [Co (NH3)4]3+ was seen bound by inner-sphere coordination to the RNA. This raises some questions about the practical use of [Co (NH3)6]3+ as a [Mg (H2O)6]2+ mimetic. Also very unexpected was the binding of the small Au3+ cation exactly between the Watson-Crick sites of a G-C base pair after an obligatory deprotonation of N1 of the guanine base. This extensive study of metal ion binding using X-ray crystallography significantly enriches our knowledge on the binding of middleweight or heavy metal ions to RNA, particularly compared with magnesium.},
note = {1362-4962
Journal Article},
keywords = {Base Sequence Binding Sites/genetics Binding, Competitive Cations, Divalent/chemistry/metabolism Cobalt/chemistry/metabolism Comparative Study Crystallization Crystallography, ENNIFAR, Molecular Nucleic Acid Heteroduplexes/*chemistry/genetics/metabolism Oligoribonucleotides/chemistry/genetics/metabolism Platinum Compounds/chemistry/metabolism RNA/*chemistry/genetics/metabolism Ruthenium Compounds/chemistry/metabolism Support, Non-U.S. Gov't, Unité ARN, X-Ray Gold Compounds/chemistry/metabolism Magnesium/chemistry/metabolism Metals/*chemistry/metabolism Models},
pubstate = {published},
tppubtype = {article}
}
Metal ions, and magnesium in particular, are known to be involved in RNA folding by stabilizing secondary and tertiary structures, and, as cofactors, in RNA enzymatic activity. We have conducted a systematic crystallographic analysis of cation binding to the duplex form of the HIV-1 RNA dimerization initiation site for the subtype-A and -B natural sequences. Eleven ions (K+, Pb2+, Mn2+, Ba2+, Ca2+, Cd2+, Sr2+, Zn2+, Co2+, Au3+ and Pt4+) and two hexammines [Co (NH3)6]3+ and [Ru (NH3)6]3+ were found to bind to the DIS duplex structure. Although the two sequences are very similar, strong differences were found in their cation binding properties. Divalent cations bind almost exclusively, as Mg2+, at 'Hoogsteen' sites of guanine residues, with a cation-dependent affinity for each site. Notably, a given cation can have very different affinities for a priori equivalent sites within the same molecule. Surprisingly, none of the two hexammines used were able to efficiently replace hexahydrated magnesium. Instead, [Co (NH3)4]3+ was seen bound by inner-sphere coordination to the RNA. This raises some questions about the practical use of [Co (NH3)6]3+ as a [Mg (H2O)6]2+ mimetic. Also very unexpected was the binding of the small Au3+ cation exactly between the Watson-Crick sites of a G-C base pair after an obligatory deprotonation of N1 of the guanine base. This extensive study of metal ion binding using X-ray crystallography significantly enriches our knowledge on the binding of middleweight or heavy metal ions to RNA, particularly compared with magnesium.
Ennifar E, Paillart J C, Marquet R, Ehresmann B, Ehresmann C, Dumas P, Walter P
HIV-1 RNA dimerization initiation site is structurally similar to the ribosomal A site and binds aminoglycoside antibiotics Article de journal
Dans: J Biol Chem, vol. 278, no. 4, p. 2723-2730, 2003, ISBN: 12435744, (0021-9258 Journal Article).
Résumé | Liens | BibTeX | Étiquettes: Anti-Bacterial Agents/*pharmacology Binding Sites Dimerization HIV-1/*metabolism Models, ENNIFAR, MARQUET, Molecular Neomycin/pharmacology Nucleic Acid Conformation Paromomycin/pharmacology Protein Binding RNA/metabolism *RNA, Non-U.S. Gov't Temperature Ultraviolet Rays, PAILLART, Unité ARN, Viral Ribosomes/*metabolism Support
@article{,
title = {HIV-1 RNA dimerization initiation site is structurally similar to the ribosomal A site and binds aminoglycoside antibiotics},
author = {E Ennifar and J C Paillart and R Marquet and B Ehresmann and C Ehresmann and P Dumas and P Walter},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12435744},
isbn = {12435744},
year = {2003},
date = {2003-01-01},
journal = {J Biol Chem},
volume = {278},
number = {4},
pages = {2723-2730},
abstract = {Human immunodeficiency virus (HIV) genomic RNA is packaged into virions as a dimer. The first step of dimerization is the formation of a kissing-loop complex at the so-called dimerization initiation site (DIS). We found an unexpected and fortuitous resemblance between the HIV-1 DIS kissing-loop complex and the eubacterial 16 S ribosomal aminoacyl-tRNA site (A site), which is the target of aminoglycoside antibiotics. Similarities exist not only at the primary and secondary structure level but also at the tertiary structure level, as revealed by comparison of the respective DIS and A site crystal structures. Gel shift, inhibition of lead-induced cleavage, and footprinting experiments showed that paromomycin and neomycin specifically bind to the kissing-loop complex formed by the DIS, with an affinity and a geometry similar to that observed for the A site. Modeling of the aminoglycoside-DIS complex allowed us to identify antibiotic modifications likely to increase the affinity and/or the specificity for the DIS. This could be a starting point for designing antiviral drugs against HIV-1 RNA dimerization.},
note = {0021-9258
Journal Article},
keywords = {Anti-Bacterial Agents/*pharmacology Binding Sites Dimerization HIV-1/*metabolism Models, ENNIFAR, MARQUET, Molecular Neomycin/pharmacology Nucleic Acid Conformation Paromomycin/pharmacology Protein Binding RNA/metabolism *RNA, Non-U.S. Gov't Temperature Ultraviolet Rays, PAILLART, Unité ARN, Viral Ribosomes/*metabolism Support},
pubstate = {published},
tppubtype = {article}
}
Human immunodeficiency virus (HIV) genomic RNA is packaged into virions as a dimer. The first step of dimerization is the formation of a kissing-loop complex at the so-called dimerization initiation site (DIS). We found an unexpected and fortuitous resemblance between the HIV-1 DIS kissing-loop complex and the eubacterial 16 S ribosomal aminoacyl-tRNA site (A site), which is the target of aminoglycoside antibiotics. Similarities exist not only at the primary and secondary structure level but also at the tertiary structure level, as revealed by comparison of the respective DIS and A site crystal structures. Gel shift, inhibition of lead-induced cleavage, and footprinting experiments showed that paromomycin and neomycin specifically bind to the kissing-loop complex formed by the DIS, with an affinity and a geometry similar to that observed for the A site. Modeling of the aminoglycoside-DIS complex allowed us to identify antibiotic modifications likely to increase the affinity and/or the specificity for the DIS. This could be a starting point for designing antiviral drugs against HIV-1 RNA dimerization.
2002
Ennifar E, Carpentier P, Ferrer J L, Walter P, Dumas P
X-ray-induced debromination of nucleic acids at the Br K absorption edge and implications for MAD phasing Article de journal
Dans: Acta Crystallogr D Biol Crystallogr, vol. 58, no. Pt 8, p. 1262-1268, 2002, ISBN: 12136136, (0907-4449 Journal Article).
Résumé | Liens | BibTeX | Étiquettes: Bromine/chemistry/radiation effects Crystallography, ENNIFAR, Non-U.S. Gov't X-Rays, Unité ARN, Viral/chemistry Support, X-Ray HIV-1/chemistry Molecular Structure Nucleic Acids/*chemistry/radiation effects RNA
@article{,
title = {X-ray-induced debromination of nucleic acids at the Br K absorption edge and implications for MAD phasing},
author = {E Ennifar and P Carpentier and J L Ferrer and P Walter and P Dumas},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12136136},
isbn = {12136136},
year = {2002},
date = {2002-01-01},
journal = {Acta Crystallogr D Biol Crystallogr},
volume = {58},
number = {Pt 8},
pages = {1262-1268},
abstract = {Multi-wavelength anomalous dispersion (MAD) using brominated derivatives is considered a common and convenient technique for solving chemically synthesized nucleic acid structures. Here, it is shown that a relatively moderate X-ray dose (of the order of 5 x 10(15) photons mm(-2)) can induce sufficient debromination to prevent structure determination. The decrease in bromine occupancy with radiation dose can be accounted for by a simple exponential, with an estimated rate constant at the absorption-peak wavelength, 7.4 (0.8) MGy, that is not significantly different from its value at the absorption-edge wavelength, 9.2 (2.6) MGy (the given e.s.d.s assess the relative closeness of the two values, not their absolute accuracy, which is probably worse). Chemically, these results (and others) are consistent with bromine cleavage resulting from direct photodissociation and/or from the action of free electrons, rather than from the action of hydroxyl radicals originating from water dissociation. The free bromine species (Br(-)) diffuse too quickly, even in amorphous ice around 100 K, to allow the determination of a diffusion coefficient. From a practical point of view, it is suggested that a single data collection with a crystal consisting of iodinated instead of brominated derivatives could provide both anomalous scattering and SIR phase information by the progressive cleavage of iodine.},
note = {0907-4449
Journal Article},
keywords = {Bromine/chemistry/radiation effects Crystallography, ENNIFAR, Non-U.S. Gov't X-Rays, Unité ARN, Viral/chemistry Support, X-Ray HIV-1/chemistry Molecular Structure Nucleic Acids/*chemistry/radiation effects RNA},
pubstate = {published},
tppubtype = {article}
}
Multi-wavelength anomalous dispersion (MAD) using brominated derivatives is considered a common and convenient technique for solving chemically synthesized nucleic acid structures. Here, it is shown that a relatively moderate X-ray dose (of the order of 5 x 10(15) photons mm(-2)) can induce sufficient debromination to prevent structure determination. The decrease in bromine occupancy with radiation dose can be accounted for by a simple exponential, with an estimated rate constant at the absorption-peak wavelength, 7.4 (0.8) MGy, that is not significantly different from its value at the absorption-edge wavelength, 9.2 (2.6) MGy (the given e.s.d.s assess the relative closeness of the two values, not their absolute accuracy, which is probably worse). Chemically, these results (and others) are consistent with bromine cleavage resulting from direct photodissociation and/or from the action of free electrons, rather than from the action of hydroxyl radicals originating from water dissociation. The free bromine species (Br(-)) diffuse too quickly, even in amorphous ice around 100 K, to allow the determination of a diffusion coefficient. From a practical point of view, it is suggested that a single data collection with a crystal consisting of iodinated instead of brominated derivatives could provide both anomalous scattering and SIR phase information by the progressive cleavage of iodine.
Serganov A, Ennifar E, Portier C, Ehresmann B, Ehresmann C
Do mRNA and rRNA binding sites of E.coli ribosomal protein S15 share common structural determinants? Article de journal
Dans: J Mol Biol, vol. 320, no. 5, p. 963-978, 2002, ISBN: 12126618, (0022-2836 Journal Article).
Résumé | Liens | BibTeX | Étiquettes: Binding Sites Cytosine Escherichia coli Guanosine Models, ENNIFAR, Messenger/*chemistry RNA, Molecular Nucleic Acid Conformation Protein Structure, Non-U.S. Gov't Uridine, Ribosomal/*chemistry Ribosomal Proteins/*chemistry Support, Tertiary RNA, Unité ARN
@article{,
title = {Do mRNA and rRNA binding sites of E.coli ribosomal protein S15 share common structural determinants?},
author = {A Serganov and E Ennifar and C Portier and B Ehresmann and C Ehresmann},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12126618},
isbn = {12126618},
year = {2002},
date = {2002-01-01},
journal = {J Mol Biol},
volume = {320},
number = {5},
pages = {963-978},
abstract = {Escherichia coli ribosomal protein S15 recognizes two RNA targets: a three-way junction in 16S rRNA and a pseudoknot structure on its own mRNA. Binding to mRNA occurs when S15 is expressed in excess over its rRNA target, resulting in an inhibition of translation start. The sole apparent similarity between the rRNA and mRNA targets is the presence of a G-U/G-C motif that contributes only modestly to rRNA binding but is essential for mRNA. To get more information on the structural determinants used by S15 to bind its mRNA target as compared to its rRNA site, we used site-directed mutagenesis, substitution by nucleotide analogs, footprinting experiments on both RNA and protein, and graphic modeling. The size of the mRNA-binding site could be reduced to 45 nucleotides, without loss of affinity. This short RNA preferentially folds into a pseudoknot, the formation of which depends on magnesium concentration and temperature. The size of the loop L2 that bridges the two stems of the pseudoknot through the minor groove could not be reduced below nine nucleotides. Then we showed that the pseudoknot recognizes the same side of S15 as 16S rRNA, although shielding a smaller surface area. It turned out that the G-U/G-C motif is recognized from the minor groove in both cases, and that the G-C pair is recognized in a very similar manner. However, the wobble G-U pair of the mRNA is not directly contacted by S15, as in rRNA, but is most likely involved in building a precise conformation of the RNA, essential for binding. Otherwise, unique specific features are utilized, such as the three-way junction in the case of 16S rRNA and the looped out A(-46) for the mRNA pseudoknot.},
note = {0022-2836
Journal Article},
keywords = {Binding Sites Cytosine Escherichia coli Guanosine Models, ENNIFAR, Messenger/*chemistry RNA, Molecular Nucleic Acid Conformation Protein Structure, Non-U.S. Gov't Uridine, Ribosomal/*chemistry Ribosomal Proteins/*chemistry Support, Tertiary RNA, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Escherichia coli ribosomal protein S15 recognizes two RNA targets: a three-way junction in 16S rRNA and a pseudoknot structure on its own mRNA. Binding to mRNA occurs when S15 is expressed in excess over its rRNA target, resulting in an inhibition of translation start. The sole apparent similarity between the rRNA and mRNA targets is the presence of a G-U/G-C motif that contributes only modestly to rRNA binding but is essential for mRNA. To get more information on the structural determinants used by S15 to bind its mRNA target as compared to its rRNA site, we used site-directed mutagenesis, substitution by nucleotide analogs, footprinting experiments on both RNA and protein, and graphic modeling. The size of the mRNA-binding site could be reduced to 45 nucleotides, without loss of affinity. This short RNA preferentially folds into a pseudoknot, the formation of which depends on magnesium concentration and temperature. The size of the loop L2 that bridges the two stems of the pseudoknot through the minor groove could not be reduced below nine nucleotides. Then we showed that the pseudoknot recognizes the same side of S15 as 16S rRNA, although shielding a smaller surface area. It turned out that the G-U/G-C motif is recognized from the minor groove in both cases, and that the G-C pair is recognized in a very similar manner. However, the wobble G-U pair of the mRNA is not directly contacted by S15, as in rRNA, but is most likely involved in building a precise conformation of the RNA, essential for binding. Otherwise, unique specific features are utilized, such as the three-way junction in the case of 16S rRNA and the looped out A(-46) for the mRNA pseudoknot.
2001
Serganov A, Benard L, Portier C, Ennifar E, Garber M, Ehresmann B, Ehresmann C
Role of conserved nucleotides in building the 16 S rRNA binding site for ribosomal protein S15 Article de journal
Dans: J Mol Biol, vol. 305, no. 4, p. 785-803, 2001, ISBN: 11162092, (0022-2836 Journal Article).
Résumé | Liens | BibTeX | Étiquettes: 16S/chemistry/*genetics/*metabolism RNA-Binding Proteins/chemistry/metabolism Ribosomal Proteins/chemistry/*metabolism Sequence Alignment Support, Amino Acid Sequence Base Pairing Base Sequence Binding Sites Conserved Sequence/*genetics *Escherichia coli/chemistry/genetics/metabolism Models, ENNIFAR, Molecular Molecular Sequence Data Mutation/genetics Nuclease Protection Assays Phylogeny Protein Binding Protein Conformation Purines/metabolism RNA, Non-U.S. Gov't Thermodynamics Thermus thermophilus/chemistry, Ribosomal, Unité ARN
@article{,
title = {Role of conserved nucleotides in building the 16 S rRNA binding site for ribosomal protein S15},
author = {A Serganov and L Benard and C Portier and E Ennifar and M Garber and B Ehresmann and C Ehresmann},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11162092},
isbn = {11162092},
year = {2001},
date = {2001-01-01},
journal = {J Mol Biol},
volume = {305},
number = {4},
pages = {785-803},
abstract = {Ribosomal protein S15 recognizes a highly conserved target on 16 S rRNA, which consists of two distinct binding regions. Here, we used extensive site-directed mutagenesis on a Escherichia coli 16 S rRNA fragment containing the S15 binding site, to investigate the role of conserved nucleotides in protein recognition and to evaluate the relative contribution of the two sites. The effect of mutations on S15 recognition was studied by measuring the relative binding affinity, RNA probing and footprinting. The crystallographic structure of the Thermus thermophilus complex allowed molecular modelling of the E. coli complex and facilitated interpretation of biochemical data. Binding is essentially driven by site 1, which includes a three-way junction constrained by a conserved base triple and cross-strand stacking. Recognition is based mainly on shape complementarity, and the role of conserved nucleotides is to maintain a unique backbone geometry. The wild-type base triple is absolutely required for protein interaction, while changes in the conserved surrounding nucleotides are partially tolerated. Site 2, which provides functional groups in a conserved G-U/G-C motif, contributes only modestly to the stability of the interaction. Binding to this motif is dependent on binding at site 1 and is allowed only if the two sites are in the correct relative orientation. Non-conserved bulged nucleotides as well as a conserved purine interior loop, although not directly involved in recognition, are used to provide an appropriate flexibility between the two sites. In addition, correct binding at the two sites triggers conformational adjustments in the purine interior loop and in a distal region, which are known to be involved for subsequent binding of proteins S6 and S18. Thus, the role of site 1 is to anchor S15 to the rRNA, while binding at site 2 is aimed to induce a cascade of events required for subunit assembly.},
note = {0022-2836
Journal Article},
keywords = {16S/chemistry/*genetics/*metabolism RNA-Binding Proteins/chemistry/metabolism Ribosomal Proteins/chemistry/*metabolism Sequence Alignment Support, Amino Acid Sequence Base Pairing Base Sequence Binding Sites Conserved Sequence/*genetics *Escherichia coli/chemistry/genetics/metabolism Models, ENNIFAR, Molecular Molecular Sequence Data Mutation/genetics Nuclease Protection Assays Phylogeny Protein Binding Protein Conformation Purines/metabolism RNA, Non-U.S. Gov't Thermodynamics Thermus thermophilus/chemistry, Ribosomal, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Ribosomal protein S15 recognizes a highly conserved target on 16 S rRNA, which consists of two distinct binding regions. Here, we used extensive site-directed mutagenesis on a Escherichia coli 16 S rRNA fragment containing the S15 binding site, to investigate the role of conserved nucleotides in protein recognition and to evaluate the relative contribution of the two sites. The effect of mutations on S15 recognition was studied by measuring the relative binding affinity, RNA probing and footprinting. The crystallographic structure of the Thermus thermophilus complex allowed molecular modelling of the E. coli complex and facilitated interpretation of biochemical data. Binding is essentially driven by site 1, which includes a three-way junction constrained by a conserved base triple and cross-strand stacking. Recognition is based mainly on shape complementarity, and the role of conserved nucleotides is to maintain a unique backbone geometry. The wild-type base triple is absolutely required for protein interaction, while changes in the conserved surrounding nucleotides are partially tolerated. Site 2, which provides functional groups in a conserved G-U/G-C motif, contributes only modestly to the stability of the interaction. Binding to this motif is dependent on binding at site 1 and is allowed only if the two sites are in the correct relative orientation. Non-conserved bulged nucleotides as well as a conserved purine interior loop, although not directly involved in recognition, are used to provide an appropriate flexibility between the two sites. In addition, correct binding at the two sites triggers conformational adjustments in the purine interior loop and in a distal region, which are known to be involved for subsequent binding of proteins S6 and S18. Thus, the role of site 1 is to anchor S15 to the rRNA, while binding at site 2 is aimed to induce a cascade of events required for subunit assembly.
2000
Nikulin A, Serganov A, Ennifar E, Tishchenko S, Nevskaya N, Shepard W, Portier C, Garber M, Ehresmann B, Ehresmann C, Nikonov S, Dumas P
Crystal structure of the S15-rRNA complex Article de journal
Dans: Nat Struct Biol, vol. 7, no. 4, p. 273-277, 2000, ISBN: 10742169, (1072-8368 Journal Article).
Résumé | Liens | BibTeX | Étiquettes: 16S/*chemistry/genetics/*metabolism Ribosomal Proteins/*chemistry/*metabolism Structure-Activity Relationship Support, Amino Acid Sequence Base Pairing/drug effects/genetics Base Sequence Binding Sites/drug effects Conserved Sequence/genetics Crystallography, Bacterial/chemistry/genetics/metabolism RNA, ENNIFAR, Molecular Molecular Sequence Data *Nucleic Acid Conformation/drug effects Protein Conformation RNA, Non-U.S. Gov't Thermus thermophilus/*chemistry/genetics, Ribosomal, Unité ARN, X-Ray Magnesium/pharmacology Models
@article{,
title = {Crystal structure of the S15-rRNA complex},
author = {A Nikulin and A Serganov and E Ennifar and S Tishchenko and N Nevskaya and W Shepard and C Portier and M Garber and B Ehresmann and C Ehresmann and S Nikonov and P Dumas},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10742169},
isbn = {10742169},
year = {2000},
date = {2000-01-01},
journal = {Nat Struct Biol},
volume = {7},
number = {4},
pages = {273-277},
abstract = {In bacterial ribosomes, the small (30S) ribosomal subunit is composed of 16S rRNA and 21 distinct proteins. Ribosomal protein S15 is of particular interest because it binds primarily to 16S rRNA and is required for assembly of the small subunit and for intersubunit association, thus representing a key element in the assembly of a whole ribosome. Here we report the 2.8 inverted question mark resolution crystal structure of the highly conserved S15-rRNA complex. Protein S15 interacts in the minor groove with a G-U/G-C motif and a three-way junction. The latter is constrained by a conserved base triple and stacking interactions, and locked into place by magnesium ions and protein side chains, mainly through interactions with the unique three-dimensional geometry of the backbone. The present structure gives insights into the dual role of S15 in ribosome assembly and translational regulation.},
note = {1072-8368
Journal Article},
keywords = {16S/*chemistry/genetics/*metabolism Ribosomal Proteins/*chemistry/*metabolism Structure-Activity Relationship Support, Amino Acid Sequence Base Pairing/drug effects/genetics Base Sequence Binding Sites/drug effects Conserved Sequence/genetics Crystallography, Bacterial/chemistry/genetics/metabolism RNA, ENNIFAR, Molecular Molecular Sequence Data *Nucleic Acid Conformation/drug effects Protein Conformation RNA, Non-U.S. Gov't Thermus thermophilus/*chemistry/genetics, Ribosomal, Unité ARN, X-Ray Magnesium/pharmacology Models},
pubstate = {published},
tppubtype = {article}
}
In bacterial ribosomes, the small (30S) ribosomal subunit is composed of 16S rRNA and 21 distinct proteins. Ribosomal protein S15 is of particular interest because it binds primarily to 16S rRNA and is required for assembly of the small subunit and for intersubunit association, thus representing a key element in the assembly of a whole ribosome. Here we report the 2.8 inverted question mark resolution crystal structure of the highly conserved S15-rRNA complex. Protein S15 interacts in the minor groove with a G-U/G-C motif and a three-way junction. The latter is constrained by a conserved base triple and stacking interactions, and locked into place by magnesium ions and protein side chains, mainly through interactions with the unique three-dimensional geometry of the backbone. The present structure gives insights into the dual role of S15 in ribosome assembly and translational regulation.
Ennifar E, Nikulin A, Tishchenko S, Serganov A, Nevskaya N, Garber M, Ehresmann B, Ehresmann C, Nikonov S, Dumas P
The crystal structure of UUCG tetraloop Article de journal
Dans: J Mol Biol, vol. 304, no. 1, p. 35-42, 2000, ISBN: 11071808, (0022-2836 Journal Article).
Résumé | Liens | BibTeX | Étiquettes: 16S/*chemistry/genetics/*metabolism Ribosomal Proteins/chemistry/*metabolism Solvents Support, Base Sequence Crystallography, Biomolecular *Nucleic Acid Conformation RNA Stability RNA, ENNIFAR, Molecular Molecular Sequence Data Motion Nuclear Magnetic Resonance, Non-U.S. Gov't Thermodynamics, Ribosomal, Unité ARN, X-Ray Hydrogen Bonding Models
@article{,
title = {The crystal structure of UUCG tetraloop},
author = {E Ennifar and A Nikulin and S Tishchenko and A Serganov and N Nevskaya and M Garber and B Ehresmann and C Ehresmann and S Nikonov and P Dumas},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11071808},
isbn = {11071808},
year = {2000},
date = {2000-01-01},
journal = {J Mol Biol},
volume = {304},
number = {1},
pages = {35-42},
abstract = {All large structured RNAs contain hairpin motifs made of a stem closed by several looped nucleotides. The most frequent loop motif is the UUCG one. This motif belongs to the tetraloop family and has the peculiarity of being highly thermodynamically stable. Here, we report the first crystal structure of two UUCG tetraloops embedded in a larger RNA-protein complex solved at 2.8 A resolution. The two loops present in the asymmetric unit are in a different crystal packing environment but, nevertheless, have an identical conformation. The observed structure is globally close to that obtained in solution by nuclear magnetic resonance. However, subtle differences point to a more detailed picture of the role played by 2'-hydroxyl groups in stabilising this tetraloop.},
note = {0022-2836
Journal Article},
keywords = {16S/*chemistry/genetics/*metabolism Ribosomal Proteins/chemistry/*metabolism Solvents Support, Base Sequence Crystallography, Biomolecular *Nucleic Acid Conformation RNA Stability RNA, ENNIFAR, Molecular Molecular Sequence Data Motion Nuclear Magnetic Resonance, Non-U.S. Gov't Thermodynamics, Ribosomal, Unité ARN, X-Ray Hydrogen Bonding Models},
pubstate = {published},
tppubtype = {article}
}
All large structured RNAs contain hairpin motifs made of a stem closed by several looped nucleotides. The most frequent loop motif is the UUCG one. This motif belongs to the tetraloop family and has the peculiarity of being highly thermodynamically stable. Here, we report the first crystal structure of two UUCG tetraloops embedded in a larger RNA-protein complex solved at 2.8 A resolution. The two loops present in the asymmetric unit are in a different crystal packing environment but, nevertheless, have an identical conformation. The observed structure is globally close to that obtained in solution by nuclear magnetic resonance. However, subtle differences point to a more detailed picture of the role played by 2'-hydroxyl groups in stabilising this tetraloop.
1999
Dumas P, Ennifar E, Walter P
Detection and treatment of twinning: an improvement and new results Article de journal
Dans: Acta Crystallogr D Biol Crystallogr, vol. 55, no. Pt 6, p. 1179-87, 1999, ISBN: 10329781, (0907-4449 Journal Article).
Résumé | Liens | BibTeX | Étiquettes: ENNIFAR, HIV-1/*genetics *Nucleic Acid Conformation RNA, Unité ARN, Viral/*chemistry
@article{,
title = {Detection and treatment of twinning: an improvement and new results},
author = {P Dumas and E Ennifar and P Walter},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10329781},
isbn = {10329781},
year = {1999},
date = {1999-01-01},
journal = {Acta Crystallogr D Biol Crystallogr},
volume = {55},
number = {Pt 6},
pages = {1179-87},
abstract = {This work deals with two aspects of the twinning problem. Firstly, an improvement of a known statistical test aimed at detecting twinning is presented and, secondly, a new parametrization of twinning is described, as well as a new method to obtain an accurate estimate of the degree of twinning. During work on crystals of the dimerization-initiation site of the HIV-1 genomic RNA, perfectly twinned crystals were obtained which were not immediately recognized as such by use of a known statistical method. This method, reminiscent of Wilson tests for the detection of centrosymmetric space groups, relies on calculation of <F2>/<F>2 or, equivalently, of <I2>/<I>2. It is shown that overlooking experimental errors may lead to erroneously large values of this index and, in turn, to ambiguous or incorrect conclusions. An immediate solution to this problem is presented. Independently, an alternative parametrization which expresses both the effect of twinning on intensities and the operation of untwinning to recover the correct intensities is proposed. A new method for estimating the degree of twinning is also presented. It is based upon maximization of the cross-correlation coefficients between intensities of all available data sets, and yields a fully analytical solution. Tests made with experimental data are quite satisfactory. It is suggested that the latter results could be used efficiently within the MIR method by allowing refinement, through one additional parameter only, of the twinning ratios of all data sets considered for phasing. Finally, the new parametrization of twinning has striking consequences in this correlation-based twinning determination: very unexpectedly, it yields a novel estimate of the 'twinning ratio' of a potentially twinned crystal which is fully independent of the data set used for the comparison.},
note = {0907-4449
Journal Article},
keywords = {ENNIFAR, HIV-1/*genetics *Nucleic Acid Conformation RNA, Unité ARN, Viral/*chemistry},
pubstate = {published},
tppubtype = {article}
}
This work deals with two aspects of the twinning problem. Firstly, an improvement of a known statistical test aimed at detecting twinning is presented and, secondly, a new parametrization of twinning is described, as well as a new method to obtain an accurate estimate of the degree of twinning. During work on crystals of the dimerization-initiation site of the HIV-1 genomic RNA, perfectly twinned crystals were obtained which were not immediately recognized as such by use of a known statistical method. This method, reminiscent of Wilson tests for the detection of centrosymmetric space groups, relies on calculation of <F2>/<F>2 or, equivalently, of <I2>/<I>2. It is shown that overlooking experimental errors may lead to erroneously large values of this index and, in turn, to ambiguous or incorrect conclusions. An immediate solution to this problem is presented. Independently, an alternative parametrization which expresses both the effect of twinning on intensities and the operation of untwinning to recover the correct intensities is proposed. A new method for estimating the degree of twinning is also presented. It is based upon maximization of the cross-correlation coefficients between intensities of all available data sets, and yields a fully analytical solution. Tests made with experimental data are quite satisfactory. It is suggested that the latter results could be used efficiently within the MIR method by allowing refinement, through one additional parameter only, of the twinning ratios of all data sets considered for phasing. Finally, the new parametrization of twinning has striking consequences in this correlation-based twinning determination: very unexpectedly, it yields a novel estimate of the 'twinning ratio' of a potentially twinned crystal which is fully independent of the data set used for the comparison.
Ennifar E, Yusupov M, Walter P, Marquet R, Ehresmann B, Ehresmann C, Dumas P
The crystal structure of the dimerization initiation site of genomic HIV-1 RNA reveals an extended duplex with two adenine bulges Article de journal
Dans: Structure, vol. 7, no. 11, p. 1439-49, 1999, ISBN: 10574792, (0969-2126 Journal Article).
Résumé | Liens | BibTeX | Étiquettes: Adenine/*chemistry Base Pair Mismatch Base Sequence Crystallography, ENNIFAR, Molecular Molecular Sequence Data *Nucleic Acid Conformation RNA, Non-U.S. Gov't, Unité ARN, Viral/*chemistry/metabolism Support, X-Ray Dimerization HIV-1/*genetics Magnesium/metabolism Magnetic Resonance Spectroscopy Manganese/metabolism Models
@article{,
title = {The crystal structure of the dimerization initiation site of genomic HIV-1 RNA reveals an extended duplex with two adenine bulges},
author = {E Ennifar and M Yusupov and P Walter and R Marquet and B Ehresmann and C Ehresmann and P Dumas},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10574792},
isbn = {10574792},
year = {1999},
date = {1999-01-01},
journal = {Structure},
volume = {7},
number = {11},
pages = {1439-49},
abstract = {BACKGROUND: An important step in retroviral replication is dimerization of the genomic RNA prior to encapsidation. Dimerization is initiated by the formation of a transient 'kissing-loop complex' that is thought to be subsequently matured into an extended duplex by the nucleocapsid protein (NCp). Although chemical probing and nuclear magnetic resonance spectroscopy have provided insight into the structure of the kissing-loop structure, no structural information concerning the extended-duplex state is available so far. RESULTS: The structure of a minimal HIV-1 RNA dimerization initiation site has been solved at 2.3 A resolution in two different space groups. It reveals a 22 base pair extended duplex with two noncanonical Watson-Crick-like G-A mismatches, each adjacent to a bulged-out adenine. The structure shows significant asymmetry in deep groove width and G-A base-pair conformations. A network of eight magnesium cations was clearly identified, one being unusually chelated by the 3' phosphate of each bulge across an extremely narrowed deep major groove. CONCLUSIONS: These crystal structures represent the putative matured form of the initial kissing-loop complex. They show the ability of this self-complementary RNA hairpin loop to acquire a more stable extended duplex structure. Both bulged adenines form a striking 'base grip' that could be a recognition signal, either in cis for another viral RNA sequence, or in trans for a protein, possibly the NCp. Magnesium binding might be important to promote and stabilize the observed extrahelical conformation of these bulges.},
note = {0969-2126
Journal Article},
keywords = {Adenine/*chemistry Base Pair Mismatch Base Sequence Crystallography, ENNIFAR, Molecular Molecular Sequence Data *Nucleic Acid Conformation RNA, Non-U.S. Gov't, Unité ARN, Viral/*chemistry/metabolism Support, X-Ray Dimerization HIV-1/*genetics Magnesium/metabolism Magnetic Resonance Spectroscopy Manganese/metabolism Models},
pubstate = {published},
tppubtype = {article}
}
BACKGROUND: An important step in retroviral replication is dimerization of the genomic RNA prior to encapsidation. Dimerization is initiated by the formation of a transient 'kissing-loop complex' that is thought to be subsequently matured into an extended duplex by the nucleocapsid protein (NCp). Although chemical probing and nuclear magnetic resonance spectroscopy have provided insight into the structure of the kissing-loop structure, no structural information concerning the extended-duplex state is available so far. RESULTS: The structure of a minimal HIV-1 RNA dimerization initiation site has been solved at 2.3 A resolution in two different space groups. It reveals a 22 base pair extended duplex with two noncanonical Watson-Crick-like G-A mismatches, each adjacent to a bulged-out adenine. The structure shows significant asymmetry in deep groove width and G-A base-pair conformations. A network of eight magnesium cations was clearly identified, one being unusually chelated by the 3' phosphate of each bulge across an extremely narrowed deep major groove. CONCLUSIONS: These crystal structures represent the putative matured form of the initial kissing-loop complex. They show the ability of this self-complementary RNA hairpin loop to acquire a more stable extended duplex structure. Both bulged adenines form a striking 'base grip' that could be a recognition signal, either in cis for another viral RNA sequence, or in trans for a protein, possibly the NCp. Magnesium binding might be important to promote and stabilize the observed extrahelical conformation of these bulges.
