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2004
Bergman N H, Lau N C, Lehnert V, Westhof E, Bartel D P
The three-dimensional architecture of the class I ligase ribozyme Journal Article
In: RNA, vol. 10, no. 2, pp. 176-184, 2004, ISBN: 14730016, (1355-8382 Journal Article).
Abstract | Links | BibTeX | Tags: Animals Cross-Linking Reagents Human Hydroxyl Radical/metabolism Light Magnesium/metabolism *Nucleic Acid Conformation RNA? WESTHOF, Catalytic/*metabolism Support, Non-U.S. Gov't Temperature, Unité ARN, WESTHOF Animals Cross-Linking Reagents Human Hydroxyl Radical/metabolism Light Magnesium/metabolism *Nucleic Acid Conformation RNA
@article{,
title = {The three-dimensional architecture of the class I ligase ribozyme},
author = {N H Bergman and N C Lau and V Lehnert and E Westhof and D P Bartel},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=14730016},
isbn = {14730016},
year = {2004},
date = {2004-01-01},
journal = {RNA},
volume = {10},
number = {2},
pages = {176-184},
abstract = {The class I ligase ribozyme catalyzes a Mg(++)-dependent RNA-ligation reaction that is chemically analogous to a single step of RNA polymerization. Indeed, this ribozyme constitutes the catalytic domain of an accurate and general RNA polymerase ribozyme. The ligation reaction is also very rapid in both single- and multiple-turnover contexts and thus is informative for the study of RNA catalysis as well as RNA self-replication. Here we report the initial characterization of the three-dimensional architecture of the ligase. When the ligase folds, several segments become protected from hydroxyl-radical cleavage, indicating that the RNA adopts a compact tertiary structure. Ribozyme folding was largely, though not completely, Mg(++) dependent, with a K(1/2[Mg]) < 1 mM, and was observed over a broad temperature range (20 degrees C -50 degrees C). The hydroxyl-radical mapping, together with comparative sequence analyses and analogy to a region within 23S ribosomal RNA, were used to generate a three-dimensional model of the ribozyme. The predictive value of the model was tested and supported by a photo-cross-linking experiment.},
note = {1355-8382
Journal Article},
keywords = {Animals Cross-Linking Reagents Human Hydroxyl Radical/metabolism Light Magnesium/metabolism *Nucleic Acid Conformation RNA? WESTHOF, Catalytic/*metabolism Support, Non-U.S. Gov't Temperature, Unité ARN, WESTHOF Animals Cross-Linking Reagents Human Hydroxyl Radical/metabolism Light Magnesium/metabolism *Nucleic Acid Conformation RNA},
pubstate = {published},
tppubtype = {article}
}
The class I ligase ribozyme catalyzes a Mg(++)-dependent RNA-ligation reaction that is chemically analogous to a single step of RNA polymerization. Indeed, this ribozyme constitutes the catalytic domain of an accurate and general RNA polymerase ribozyme. The ligation reaction is also very rapid in both single- and multiple-turnover contexts and thus is informative for the study of RNA catalysis as well as RNA self-replication. Here we report the initial characterization of the three-dimensional architecture of the ligase. When the ligase folds, several segments become protected from hydroxyl-radical cleavage, indicating that the RNA adopts a compact tertiary structure. Ribozyme folding was largely, though not completely, Mg(++) dependent, with a K(1/2[Mg]) < 1 mM, and was observed over a broad temperature range (20 degrees C -50 degrees C). The hydroxyl-radical mapping, together with comparative sequence analyses and analogy to a region within 23S ribosomal RNA, were used to generate a three-dimensional model of the ribozyme. The predictive value of the model was tested and supported by a photo-cross-linking experiment.
1997
Ng J D, Kuznetsov Y G, Malkin A J, Keith G, Giege R, McPherson A
Visualization of RNA crystal growth by atomic force microscopy Journal Article
In: Nucleic Acids Res, vol. 25, no. 13, pp. 2582-2588, 1997, ISBN: 9185567, (0305-1048 Journal Article).
Abstract | Links | BibTeX | Tags: Atomic Force RNA, Crystallization *Microscopy, Fungal/*chemistry RNA, Non-U.S. Gov't Temperature, Phe/*chemistry Saccharomyces cerevisiae/genetics Support, Transfer, Unité ARN
@article{,
title = {Visualization of RNA crystal growth by atomic force microscopy},
author = {J D Ng and Y G Kuznetsov and A J Malkin and G Keith and R Giege and A McPherson},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9185567},
isbn = {9185567},
year = {1997},
date = {1997-01-01},
journal = {Nucleic Acids Res},
volume = {25},
number = {13},
pages = {2582-2588},
abstract = {The crystallization of transfer RNA (tRNA) was investigated using atomic force microscopy (AFM) over the temperature range from 4 to 16 degrees C, and this produced the first in situ AFM images of developing nucleic acid crystals. The growth of the (110) face of hexagonal yeast tRNAPhe crystals was observed to occur at steps on vicinal hillocks generated by multiple screw dislocation sources in the temperature range of 13.5-16 degrees C. Two-dimensional nucleation begins to dominate at 13.5 degrees C, with the appearance of three-dimensional nuclei at 12 degrees C. The changes in growth mechanisms are correlated with variations in supersaturation which is higher in the low temperature range. Growth of tRNA crystals was characterized by a strong anisotropy in the tangential step movement and transformation of growth modes on single crystals were directly observed by AFM over the narrow temperature range utilized. Finally, lattice resolution images of the molecular structure of surface layers were recorded. The implications of the strong temperature dependence of tRNAPhe crystal growth are discussed in view of improving and better controlling crystallization of nucleic acids.},
note = {0305-1048
Journal Article},
keywords = {Atomic Force RNA, Crystallization *Microscopy, Fungal/*chemistry RNA, Non-U.S. Gov't Temperature, Phe/*chemistry Saccharomyces cerevisiae/genetics Support, Transfer, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The crystallization of transfer RNA (tRNA) was investigated using atomic force microscopy (AFM) over the temperature range from 4 to 16 degrees C, and this produced the first in situ AFM images of developing nucleic acid crystals. The growth of the (110) face of hexagonal yeast tRNAPhe crystals was observed to occur at steps on vicinal hillocks generated by multiple screw dislocation sources in the temperature range of 13.5-16 degrees C. Two-dimensional nucleation begins to dominate at 13.5 degrees C, with the appearance of three-dimensional nuclei at 12 degrees C. The changes in growth mechanisms are correlated with variations in supersaturation which is higher in the low temperature range. Growth of tRNA crystals was characterized by a strong anisotropy in the tangential step movement and transformation of growth modes on single crystals were directly observed by AFM over the narrow temperature range utilized. Finally, lattice resolution images of the molecular structure of surface layers were recorded. The implications of the strong temperature dependence of tRNAPhe crystal growth are discussed in view of improving and better controlling crystallization of nucleic acids.
Martin F, Sharples G J, Lloyd R G, Eiler S, Moras D, Gangloff J, Eriani G
Characterization of a thermosensitive Escherichia coli aspartyl-tRNA synthetase mutant Journal Article
In: J Bacteriol, vol. 179, no. 11, pp. 3691-3696, 1997, ISBN: 9171418, (0021-9193 Journal Article).
Abstract | Links | BibTeX | Tags: Aspartate-tRNA Ligase/*genetics Escherichia coli/*genetics Models, ERIANI, Molecular Mutation Structure-Activity Relationship Support, Non-U.S. Gov't Temperature, Unité ARN
@article{,
title = {Characterization of a thermosensitive Escherichia coli aspartyl-tRNA synthetase mutant},
author = {F Martin and G J Sharples and R G Lloyd and S Eiler and D Moras and J Gangloff and G Eriani},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9171418},
isbn = {9171418},
year = {1997},
date = {1997-01-01},
journal = {J Bacteriol},
volume = {179},
number = {11},
pages = {3691-3696},
abstract = {The Escherichia coli tls-1 strain carrying a mutated aspS gene (coding for aspartyl-tRNA synthetase), which causes a temperature-sensitive growth phenotype, was cloned by PCR, sequenced, and shown to contain a single mutation resulting in substitution by serine of the highly conserved proline 555, which is located in motif 3. When an aspS fragment spanning the codon for proline 555 was transformed into the tls-1 strain, it was shown to restore the wild-type phenotype via homologous recombination with the chromosomal tls-1 allele. The mutated AspRS purified from an overproducing strain displayed marked temperature sensitivity, with half-life values of 22 and 68 min (at 42 degrees C), respectively, for tRNA aminoacylation and ATP/PPi exchange activities. Km values for aspartic acid, ATP, and tRNA(Asp) did not significantly differ from those of the native enzyme; thus, mutation Pro555Ser lowers the stability of the functional configuration of both the acylation and the amino acid activation sites but has no significant effect on substrate binding. This decrease in stability appears to be related to a conformational change, as shown by gel filtration analysis. Structural data strongly suggest that the Pro555Ser mutation lowers the stability of the Lys556 and Thr557 positions, since these two residues, as shown by the crystallographic structure of the enzyme, are involved in the active site and in contacts with the tRNA acceptor arm, respectively.},
note = {0021-9193
Journal Article},
keywords = {Aspartate-tRNA Ligase/*genetics Escherichia coli/*genetics Models, ERIANI, Molecular Mutation Structure-Activity Relationship Support, Non-U.S. Gov't Temperature, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The Escherichia coli tls-1 strain carrying a mutated aspS gene (coding for aspartyl-tRNA synthetase), which causes a temperature-sensitive growth phenotype, was cloned by PCR, sequenced, and shown to contain a single mutation resulting in substitution by serine of the highly conserved proline 555, which is located in motif 3. When an aspS fragment spanning the codon for proline 555 was transformed into the tls-1 strain, it was shown to restore the wild-type phenotype via homologous recombination with the chromosomal tls-1 allele. The mutated AspRS purified from an overproducing strain displayed marked temperature sensitivity, with half-life values of 22 and 68 min (at 42 degrees C), respectively, for tRNA aminoacylation and ATP/PPi exchange activities. Km values for aspartic acid, ATP, and tRNA(Asp) did not significantly differ from those of the native enzyme; thus, mutation Pro555Ser lowers the stability of the functional configuration of both the acylation and the amino acid activation sites but has no significant effect on substrate binding. This decrease in stability appears to be related to a conformational change, as shown by gel filtration analysis. Structural data strongly suggest that the Pro555Ser mutation lowers the stability of the Lys556 and Thr557 positions, since these two residues, as shown by the crystallographic structure of the enzyme, are involved in the active site and in contacts with the tRNA acceptor arm, respectively.
1993
Puglisi J D, Putz J, Florentz C, Giege R
Influence of tRNA tertiary structure and stability on aminoacylation by yeast aspartyl-tRNA synthetase Journal Article
In: Nucleic Acids Res, vol. 21, no. 1, pp. 41-49, 1993, ISBN: 8441619, (0305-1048 Journal Article).
Abstract | Links | BibTeX | Tags: Acylation Aspartate-tRNA Ligase/*metabolism Base Sequence Kinetics Molecular Sequence Data Mutation *Nucleic Acid Conformation RNA, FLORENTZ, Non-U.S. Gov't Temperature, Transfer/*chemistry/metabolism Saccharomyces cerevisiae/enzymology Support, Unité ARN
@article{,
title = {Influence of tRNA tertiary structure and stability on aminoacylation by yeast aspartyl-tRNA synthetase},
author = {J D Puglisi and J Putz and C Florentz and R Giege},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8441619},
isbn = {8441619},
year = {1993},
date = {1993-01-01},
journal = {Nucleic Acids Res},
volume = {21},
number = {1},
pages = {41-49},
abstract = {Mutations have been designed that disrupt the tertiary structure of yeast tRNA(Asp). The effects of these mutations on both tRNA structure and specific aspartylation by yeast aspartyl-tRNA synthetase were assayed. Mutations that disrupt tertiary interactions involving the D-stem or D-loop result in destabilization of the base-pairing in the D-stem, as monitored by nuclease digestion and chemical modification studies. These mutations also decrease the specificity constant (kcat/Km) for aspartylation by aspartyl-tRNA synthetase up to 10(3)-10(4) fold. The size of the T-loop also influences tRNA(Asp) structure and function; change of its T-loop to a tetraloop (-UUCG-) sequence results in a denatured D-stem and an almost 10(4) fold decrease of kcat/Km for aspartylation. The negative effects of these mutations on aspartylation activity are significantly alleviated by additional mutations that stabilize the D-stem. These results indicate that a critical role of tertiary structure in tRNA(Asp) for aspartylation is the maintenance of a base-paired D-stem.},
note = {0305-1048
Journal Article},
keywords = {Acylation Aspartate-tRNA Ligase/*metabolism Base Sequence Kinetics Molecular Sequence Data Mutation *Nucleic Acid Conformation RNA, FLORENTZ, Non-U.S. Gov't Temperature, Transfer/*chemistry/metabolism Saccharomyces cerevisiae/enzymology Support, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Mutations have been designed that disrupt the tertiary structure of yeast tRNA(Asp). The effects of these mutations on both tRNA structure and specific aspartylation by yeast aspartyl-tRNA synthetase were assayed. Mutations that disrupt tertiary interactions involving the D-stem or D-loop result in destabilization of the base-pairing in the D-stem, as monitored by nuclease digestion and chemical modification studies. These mutations also decrease the specificity constant (kcat/Km) for aspartylation by aspartyl-tRNA synthetase up to 10(3)-10(4) fold. The size of the T-loop also influences tRNA(Asp) structure and function; change of its T-loop to a tetraloop (-UUCG-) sequence results in a denatured D-stem and an almost 10(4) fold decrease of kcat/Km for aspartylation. The negative effects of these mutations on aspartylation activity are significantly alleviated by additional mutations that stabilize the D-stem. These results indicate that a critical role of tertiary structure in tRNA(Asp) for aspartylation is the maintenance of a base-paired D-stem.
