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2004
Martineau Y., Bec C. Le, Monbrun L., Allo V., Chiu I. M., Danos O., Moine H., Prats H., Prats A. C.
Internal ribosome entry site structural motifs conserved among mammalian fibroblast growth factor 1 alternatively spliced mRNAs Journal Article
In: Mol Cell Biol, vol. 24, no. 17, pp. 7622-35, 2004, (0270-7306 Journal Article).
Abstract | BibTeX | Tags: (Genetics), *5', *Alternative, *Nucleic, *Promoter, 1/*genetics, Acid, Alignment, Animals, Base, Cell, Conformation, Data, EHRESMANN, Factor, Fibroblast, Gene, Genes, Genetic, Gov't, Growth, Human, Line, Messenger/chemistry/*genetics/metabolism, Mice, Molecular, Muscle, Mutagenesis, Non-U.S., Regions, Ribosomes/*metabolism, RNA, Sequence, Site-Directed, Skeletal/cytology/physiology, Splicing, Structural/genetics, Support, Techniques, Transfer, Untranslated, Vectors
@article{,
title = {Internal ribosome entry site structural motifs conserved among mammalian fibroblast growth factor 1 alternatively spliced mRNAs},
author = { Y. Martineau and C. Le Bec and L. Monbrun and V. Allo and I. M. Chiu and O. Danos and H. Moine and H. Prats and A. C. Prats},
year = {2004},
date = {2004-01-01},
journal = {Mol Cell Biol},
volume = {24},
number = {17},
pages = {7622-35},
abstract = {Fibroblast growth factor 1 (FGF-1) is a powerful angiogenic factor whose gene structure contains four promoters, giving rise to a process of alternative splicing resulting in four mRNAs with alternative 5' untranslated regions (5' UTRs). Here we have identified, by using double luciferase bicistronic vectors, the presence of internal ribosome entry sites (IRESs) in the human FGF-1 5' UTRs, particularly in leaders A and C, with distinct activities in mammalian cells. DNA electrotransfer in mouse muscle revealed that the IRES present in the FGF-1 leader A has a high activity in vivo. We have developed a new regulatable TET OFF bicistronic system, which allowed us to rule out the possibility of any cryptic promoter in the FGF-1 leaders. FGF-1 IRESs A and C, which were mapped in fragments of 118 and 103 nucleotides, respectively, are flexible in regard to the position of the initiation codon, making them interesting from a biotechnological point of view. Furthermore, we show that FGF-1 IRESs A of murine and human origins show similar IRES activity profiles. Enzymatic and chemical probing of the FGF-1 IRES A RNA revealed a structural domain conserved among mammals at both the nucleotide sequence and RNA structure levels. The functional role of this structural motif has been demonstrated by point mutagenesis, including compensatory mutations. These data favor an important role of IRESs in the control of FGF-1 expression and provide a new IRES structural motif that could help IRES prediction in 5' UTR databases.},
note = {0270-7306
Journal Article},
keywords = {(Genetics), *5', *Alternative, *Nucleic, *Promoter, 1/*genetics, Acid, Alignment, Animals, Base, Cell, Conformation, Data, EHRESMANN, Factor, Fibroblast, Gene, Genes, Genetic, Gov't, Growth, Human, Line, Messenger/chemistry/*genetics/metabolism, Mice, Molecular, Muscle, Mutagenesis, Non-U.S., Regions, Ribosomes/*metabolism, RNA, Sequence, Site-Directed, Skeletal/cytology/physiology, Splicing, Structural/genetics, Support, Techniques, Transfer, Untranslated, Vectors},
pubstate = {published},
tppubtype = {article}
}
Fibroblast growth factor 1 (FGF-1) is a powerful angiogenic factor whose gene structure contains four promoters, giving rise to a process of alternative splicing resulting in four mRNAs with alternative 5' untranslated regions (5' UTRs). Here we have identified, by using double luciferase bicistronic vectors, the presence of internal ribosome entry sites (IRESs) in the human FGF-1 5' UTRs, particularly in leaders A and C, with distinct activities in mammalian cells. DNA electrotransfer in mouse muscle revealed that the IRES present in the FGF-1 leader A has a high activity in vivo. We have developed a new regulatable TET OFF bicistronic system, which allowed us to rule out the possibility of any cryptic promoter in the FGF-1 leaders. FGF-1 IRESs A and C, which were mapped in fragments of 118 and 103 nucleotides, respectively, are flexible in regard to the position of the initiation codon, making them interesting from a biotechnological point of view. Furthermore, we show that FGF-1 IRESs A of murine and human origins show similar IRES activity profiles. Enzymatic and chemical probing of the FGF-1 IRES A RNA revealed a structural domain conserved among mammals at both the nucleotide sequence and RNA structure levels. The functional role of this structural motif has been demonstrated by point mutagenesis, including compensatory mutations. These data favor an important role of IRESs in the control of FGF-1 expression and provide a new IRES structural motif that could help IRES prediction in 5' UTR databases.
2001
Wilhelm M., Wilhelm F. X.
Reverse transcription of retroviruses and LTR retrotransposons Journal Article
In: Cell Mol Life Sci, vol. 58, no. 9, pp. 1246-62, 2001, (1420-682x Journal Article Review Review, Academic).
Abstract | BibTeX | Tags: *Retroelements, *Terminal, Acid, Alignment, Amino, Animals, Base, Conformation, Data, DNA, Homology, Human, Molecular, Nucleic, Polymerase/*chemistry/*metabolism, Repeat, Retroviridae/*enzymology/*genetics, RNA-Directed, Sequence, Sequences
@article{,
title = {Reverse transcription of retroviruses and LTR retrotransposons},
author = { M. Wilhelm and F. X. Wilhelm},
year = {2001},
date = {2001-01-01},
journal = {Cell Mol Life Sci},
volume = {58},
number = {9},
pages = {1246-62},
abstract = {Retroelements are mobile genetic entities that replicate via reverse transcription of a template RNA. A key component to the life cycle of these elements is the enzyme reverse transcriptase (RT), which copies the single-stranded genomic RNA of the element into a linear double-stranded DNA that is ultimately integrated into the host genome by the element-encoded integrase. RT is a multifunctionnal enzyme which possesses RNA-dependent and DNA-dependent DNA polymerase activities as well as RNase H activity that specifically degrades the RNA strand of RNA-DNA duplexes. At some stages of the replication a strand-displacement activity of RT is also necessary. All activities are essential for the conversion of single-stranded genomic RNA into the double-stranded preintegrative DNA. This review focuses on the role of RT in the different steps of the replication process of retroelements. The features of retrotransposon replication which differ from the retroviral ones will be emphasized. In a second part of the review, the biochemical and enzymatic properties of two newly characterized retrotransposon RTs will be described. The role of the integrase domain in reverse transcriptase activity of some retroviral and retrotransposon RTs will be discussed.},
note = {1420-682x
Journal Article
Review
Review, Academic},
keywords = {*Retroelements, *Terminal, Acid, Alignment, Amino, Animals, Base, Conformation, Data, DNA, Homology, Human, Molecular, Nucleic, Polymerase/*chemistry/*metabolism, Repeat, Retroviridae/*enzymology/*genetics, RNA-Directed, Sequence, Sequences},
pubstate = {published},
tppubtype = {article}
}
Retroelements are mobile genetic entities that replicate via reverse transcription of a template RNA. A key component to the life cycle of these elements is the enzyme reverse transcriptase (RT), which copies the single-stranded genomic RNA of the element into a linear double-stranded DNA that is ultimately integrated into the host genome by the element-encoded integrase. RT is a multifunctionnal enzyme which possesses RNA-dependent and DNA-dependent DNA polymerase activities as well as RNase H activity that specifically degrades the RNA strand of RNA-DNA duplexes. At some stages of the replication a strand-displacement activity of RT is also necessary. All activities are essential for the conversion of single-stranded genomic RNA into the double-stranded preintegrative DNA. This review focuses on the role of RT in the different steps of the replication process of retroelements. The features of retrotransposon replication which differ from the retroviral ones will be emphasized. In a second part of the review, the biochemical and enzymatic properties of two newly characterized retrotransposon RTs will be described. The role of the integrase domain in reverse transcriptase activity of some retroviral and retrotransposon RTs will be discussed.
2000
Wilhelm M., Boutabout M., Wilhelm F. X.
Expression of an active form of recombinant Ty1 reverse transcriptase in Escherichia coli: a fusion protein containing the C-terminal region of the Ty1 integrase linked to the reverse transcriptase-RNase H domain exhibits polymerase and RNase H activities Journal Article
In: Biochem J, vol. 348, no. Pt 2, pp. 337-42, 2000, (0264-6021 Journal Article).
Abstract | BibTeX | Tags: &, Acid, affinity, Alignment, Amino, Calf, cerevisiae/*enzymology/*genetics, Chromatography, Cloning, Codon, coli, Comparative, Data, DNA, DNA/metabolism, Escherichia, Frames, Fusion, Genetic, Gov't, H, Heteroduplexes/metabolism, HIV-1, Homology, Integrases/chemistry/metabolism, Kinetics, Molecular, Non-U.S., Nucleic, Open, Polymerase/chemistry/isolation, Proteins/chemistry/isolation, purification/*metabolism, purification/metabolism, Reading, Recombinant, Retroelements/*genetics, Reverse, Ribonuclease, RNA-Directed, RNA/metabolism, Saccharomyces, Sequence, Study, Support, Templates, Terminator, Thymus/isolation, Transcriptase/chemistry
@article{,
title = {Expression of an active form of recombinant Ty1 reverse transcriptase in Escherichia coli: a fusion protein containing the C-terminal region of the Ty1 integrase linked to the reverse transcriptase-RNase H domain exhibits polymerase and RNase H activities},
author = { M. Wilhelm and M. Boutabout and F. X. Wilhelm},
year = {2000},
date = {2000-01-01},
journal = {Biochem J},
volume = {348},
number = {Pt 2},
pages = {337-42},
abstract = {Replication of the Saccharomyces cerevisiae Ty1 retrotransposon requires a reverse transcriptase capable of synthesizing Ty1 DNA. The first description of an active form of a recombinant Ty1 enzyme with polymerase and RNase H activities is reported here. The Ty1 enzyme was expressed as a hexahistidine-tagged fusion protein in Escherichia coli to facilitate purification of the recombinant protein by metal-chelate chromatography. Catalytic activity of the recombinant protein was detected only when amino acid residues encoded by the integrase gene were added to the N-terminus of the reverse transcriptase-RNase H domain. This suggests that the integrase domain could play a role in proper folding of reverse transcriptase. Several biochemical properties of the Ty1 enzyme were analysed, including the effect of MgCl(2), NaCl, temperature and of the chain terminator dideoxy GTP on its polymerase activity. RNase H activity was examined by monitoring the cleavage of a RNA-DNA template-primer. Our results suggest that the distance between the RNase H and polymerase active sites corresponds to the length of a 14-nucleotide RNA-DNA heteroduplex. The recombinant protein produced in E. coli should be useful for further biochemical and structural analyses and for a better understanding of the role of integrase in the activation of reverse transcriptase.},
note = {0264-6021
Journal Article},
keywords = {&, Acid, affinity, Alignment, Amino, Calf, cerevisiae/*enzymology/*genetics, Chromatography, Cloning, Codon, coli, Comparative, Data, DNA, DNA/metabolism, Escherichia, Frames, Fusion, Genetic, Gov't, H, Heteroduplexes/metabolism, HIV-1, Homology, Integrases/chemistry/metabolism, Kinetics, Molecular, Non-U.S., Nucleic, Open, Polymerase/chemistry/isolation, Proteins/chemistry/isolation, purification/*metabolism, purification/metabolism, Reading, Recombinant, Retroelements/*genetics, Reverse, Ribonuclease, RNA-Directed, RNA/metabolism, Saccharomyces, Sequence, Study, Support, Templates, Terminator, Thymus/isolation, Transcriptase/chemistry},
pubstate = {published},
tppubtype = {article}
}
Replication of the Saccharomyces cerevisiae Ty1 retrotransposon requires a reverse transcriptase capable of synthesizing Ty1 DNA. The first description of an active form of a recombinant Ty1 enzyme with polymerase and RNase H activities is reported here. The Ty1 enzyme was expressed as a hexahistidine-tagged fusion protein in Escherichia coli to facilitate purification of the recombinant protein by metal-chelate chromatography. Catalytic activity of the recombinant protein was detected only when amino acid residues encoded by the integrase gene were added to the N-terminus of the reverse transcriptase-RNase H domain. This suggests that the integrase domain could play a role in proper folding of reverse transcriptase. Several biochemical properties of the Ty1 enzyme were analysed, including the effect of MgCl(2), NaCl, temperature and of the chain terminator dideoxy GTP on its polymerase activity. RNase H activity was examined by monitoring the cleavage of a RNA-DNA template-primer. Our results suggest that the distance between the RNase H and polymerase active sites corresponds to the length of a 14-nucleotide RNA-DNA heteroduplex. The recombinant protein produced in E. coli should be useful for further biochemical and structural analyses and for a better understanding of the role of integrase in the activation of reverse transcriptase.
1997
Bergdoll M., Remy M. H., Cagnon C., Masson J. M., Dumas P.
Proline-dependent oligomerization with arm exchange Journal Article
In: Structure, vol. 5, no. 3, pp. 391-401, 1997, (0969-2126 Journal Article).
Abstract | BibTeX | Tags: *Acetyltransferases, *Dimerization, *Protein, Acid, Alignment, Amino, Aminotransferases/chemistry, Animals, Aspartate, ATPase/chemistry, Bacterial, Binding, Cattle, Chickens, Comparative, Conformation, Data, Folding, Heart/enzymology, Human, mitochondria, Models, Molecular, Mutagenesis, Na(+)-K(+)-Exchanging, Pancreatic/chemistry, Plant, Proline/*physiology, Protein, Proteins/chemistry, Pyrophosphatases/chemistry, Ribonuclease, Sequence, Site-Directed, Structural, Study, Viral, Viruses/chemistry
@article{,
title = {Proline-dependent oligomerization with arm exchange},
author = { M. Bergdoll and M. H. Remy and C. Cagnon and J. M. Masson and P. Dumas},
year = {1997},
date = {1997-01-01},
journal = {Structure},
volume = {5},
number = {3},
pages = {391-401},
abstract = {BACKGROUND: Oligomerization is often necessary for protein activity or regulation and its efficiency is fundamental for the cell. The quaternary structure of a large number of oligomers consists of protomers tightly anchored to each other by exchanged arms or swapped domains. However, nothing is known about how the arms can be kept in a favourable conformation before such an oligomerization. RESULTS: Upon examination of such quaternary structures, we observe an extremely frequent occurrence of proline residues at the point where the arm leaves the protomer. Sequence alignment and site-directed mutagenesis confirm the importance of these prolines. The conservation of these residues at the hinge regions can be explained by the constraints that they impose on polypeptide conformation and dynamics: by rigidifying the mainchain, prolines favour extended conformations of arms thus favouring oligomerization, and may prevent interaction of the arms with the core of the protomer. CONCLUSIONS: Hinge prolines can be considered as 'quaternary structure helpers'. The presence of a proline should be considered when searching for a determinant of oligomerization with arm exchange and could be used to engineer synthetic oligomers or to displace a monomers to oligomers equilibrium by mutation of this proline residue.},
note = {0969-2126
Journal Article},
keywords = {*Acetyltransferases, *Dimerization, *Protein, Acid, Alignment, Amino, Aminotransferases/chemistry, Animals, Aspartate, ATPase/chemistry, Bacterial, Binding, Cattle, Chickens, Comparative, Conformation, Data, Folding, Heart/enzymology, Human, mitochondria, Models, Molecular, Mutagenesis, Na(+)-K(+)-Exchanging, Pancreatic/chemistry, Plant, Proline/*physiology, Protein, Proteins/chemistry, Pyrophosphatases/chemistry, Ribonuclease, Sequence, Site-Directed, Structural, Study, Viral, Viruses/chemistry},
pubstate = {published},
tppubtype = {article}
}
BACKGROUND: Oligomerization is often necessary for protein activity or regulation and its efficiency is fundamental for the cell. The quaternary structure of a large number of oligomers consists of protomers tightly anchored to each other by exchanged arms or swapped domains. However, nothing is known about how the arms can be kept in a favourable conformation before such an oligomerization. RESULTS: Upon examination of such quaternary structures, we observe an extremely frequent occurrence of proline residues at the point where the arm leaves the protomer. Sequence alignment and site-directed mutagenesis confirm the importance of these prolines. The conservation of these residues at the hinge regions can be explained by the constraints that they impose on polypeptide conformation and dynamics: by rigidifying the mainchain, prolines favour extended conformations of arms thus favouring oligomerization, and may prevent interaction of the arms with the core of the protomer. CONCLUSIONS: Hinge prolines can be considered as 'quaternary structure helpers'. The presence of a proline should be considered when searching for a determinant of oligomerization with arm exchange and could be used to engineer synthetic oligomers or to displace a monomers to oligomers equilibrium by mutation of this proline residue.
Friant S., Heyman T., Poch O., Wilhelm M., Wilhelm F. X.
Sequence comparison of the Ty1 and Ty2 elements of the yeast genome supports the structural model of the tRNAiMet-Ty1 RNA reverse transcription initiation complex Journal Article
In: Yeast, vol. 13, no. 7, pp. 639-45, 1997, (0749-503x Journal Article).
Abstract | BibTeX | Tags: *Sequence, Acid, Alignment, Amino, Analysis, Base, Data, DNA, Elements/*genetics, Fungal/genetics, Gov't, Met/*chemistry/*genetics, Molecular, Non-U.S., RNA, Sequence, structure, Support, Transfer, Transposable, Yeasts/*genetics
@article{,
title = {Sequence comparison of the Ty1 and Ty2 elements of the yeast genome supports the structural model of the tRNAiMet-Ty1 RNA reverse transcription initiation complex},
author = { S. Friant and T. Heyman and O. Poch and M. Wilhelm and F. X. Wilhelm},
year = {1997},
date = {1997-01-01},
journal = {Yeast},
volume = {13},
number = {7},
pages = {639-45},
abstract = {In the reverse transcription initiation complex of the yeast Ty1 retrotransposon, interaction between the template RNA and primer tRNAiMet is not limited to base pairing of the primer binding site (PBS) with ten nucleotides at the 3' end of tRNAiMet, but three regions named boxes O, 1 and 2.1 interact with the T and D stems and loops of tRNAiMet. Sequence comparison of 33 Ty1 elements and 13 closely related Ty2 elements found in the yeast genome shows that the nucleotide sequence of all elements is highly conserved in the region spanning the PBS and the three boxes. Since the domain of the template RNA encodes a portion of protein TyA, we have calculated its amino acid profile and its nucleotide profile to evaluate the role played by nucleotide sequence conservation in the selection for TyA function and in the maintenance of base pairing interactions for the priming function of Ty1 RNA. Our results show that the nucleotide sequence conservation of Ty1 RNA is constrained not only by selection for Ty1 function but also by maintenance of a given nucleotide sequence able to base pair with the tRNAiMet in the primer-template initiation complex.},
note = {0749-503x
Journal Article},
keywords = {*Sequence, Acid, Alignment, Amino, Analysis, Base, Data, DNA, Elements/*genetics, Fungal/genetics, Gov't, Met/*chemistry/*genetics, Molecular, Non-U.S., RNA, Sequence, structure, Support, Transfer, Transposable, Yeasts/*genetics},
pubstate = {published},
tppubtype = {article}
}
In the reverse transcription initiation complex of the yeast Ty1 retrotransposon, interaction between the template RNA and primer tRNAiMet is not limited to base pairing of the primer binding site (PBS) with ten nucleotides at the 3' end of tRNAiMet, but three regions named boxes O, 1 and 2.1 interact with the T and D stems and loops of tRNAiMet. Sequence comparison of 33 Ty1 elements and 13 closely related Ty2 elements found in the yeast genome shows that the nucleotide sequence of all elements is highly conserved in the region spanning the PBS and the three boxes. Since the domain of the template RNA encodes a portion of protein TyA, we have calculated its amino acid profile and its nucleotide profile to evaluate the role played by nucleotide sequence conservation in the selection for TyA function and in the maintenance of base pairing interactions for the priming function of Ty1 RNA. Our results show that the nucleotide sequence conservation of Ty1 RNA is constrained not only by selection for Ty1 function but also by maintenance of a given nucleotide sequence able to base pair with the tRNAiMet in the primer-template initiation complex.
