Selection of suppressor methionyl-tRNA synthetases: mapping the tRNA anticodon binding site.

Meinnel, Thierry; Mechulam, Yves; Corre, Daniel Le; Panvert, Michel; Blanquet, Sylvain; Fayat, Guy

doi:10.1073/pnas.88.1.291

Cited by 74 publications

(67 citation statements)

References 27 publications

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“…Extensive experiments have been carried out to establish the importance of several amino acid residues in MetRS and the nucleotides of tRNA in the recognition processes. For instance, the loop containing highly conserved residue Trp-461 plays a crucial role in the recognition of anticodon bases, as confirmed by affinity-labeling experiments (5), isolation of second site mutants (6), and docking studies of glutaminyl-tRNA on MetRS (7). Substitution of highly conserved residues Trp-461, Asn-452, or Arg-395 shows their involvement in the binding of MetRS to anticodon site of tRNA (8)(9)(10).…”

mentioning

confidence: 99%

A study of communication pathways in methionyl- tRNA synthetase by molecular dynamics simulations and structure network analysis

Ghosh

Vishveshwara

2007

Proc. Natl. Acad. Sci. U.S.A.

229

288

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The enzymes of the family of tRNA synthetases perform their functions with high precision by synchronously recognizing the anticodon region and the aminoacylation region, which are separated by Ϸ70 Å in space. This precision in function is brought about by establishing good communication paths between the two regions. We have modeled the structure of the complex consisting of Escherichia coli methionyl-tRNA synthetase (MetRS), tRNA, and the activated methionine. Molecular dynamics simulations have been performed on the modeled structure to obtain the equilibrated structure of the complex and the cross-correlations between the residues in MetRS have been evaluated. Furthermore, the network analysis on these simulated structures has been carried out to elucidate the paths of communication between the activation site and the anticodon recognition site. This study has provided the detailed paths of communication, which are consistent with experimental results. Similar studies also have been carried out on the complexes (MetRS ؉ activated methonine) and (MetRS ؉ tRNA) along with ligand-free native enzyme. A comparison of the paths derived from the four simulations clearly has shown that the communication path is strongly correlated and unique to the enzyme complex, which is bound to both the tRNA and the activated methionine. The details of the method of our investigation and the biological implications of the results are presented in this article. The method developed here also could be used to investigate any protein system where the function takes place through longdistance communication.dynamic cross-correlations ͉ methionyl-AMP ͉ protein structure network ͉ shortest pathways of communication ͉ stacking A crucial step in the translation of the genetic code is the aminoacylation of tRNA, which involves the molecular recognition between the aminoacyl-tRNA synthetases (aaRS) and their cognate tRNA. Each synthetase consists of the catalytic domain and the anticodon domain that are separated by Ϸ70 Å. Each tRNA connects these two regions with its anticodon and the acceptor stems. The mechanism of differentiations between cognate and noncognate tRNAs depends on contacts of anticodon domain of synthetase and anticodon stem of tRNA. The efficiency of the selection mechanism controls the overall accuracy of protein synthesis (1, 2). Recognition of the protein (aaRS) and the tRNA is explained by using the induced-fit mechanism, which suggests conformational changes in protein, tRNA, or both, leading to the final bound complex (3). However, the details of communication between the anticodon region and the aminoacylation region are less understood.In all living cells, protein synthesis starts with methionine.

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mentioning

confidence: 99%

A study of communication pathways in methionyl- tRNA synthetase by molecular dynamics simulations and structure network analysis

Ghosh

Vishveshwara

2007

Proc. Natl. Acad. Sci. U.S.A.

229

288

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“…The mutant genes were cloned in specialized pUCl2-derived expression vectors (Mechulam et al, 1991 b) and expressed in JMlOlTr cells (Hire1 et al, 1988) in the presence of 0.3 mM isopropyl b-D-thiogalactoside. B. stenrothermophilus MS.534 variants were then purified to homogeneity using a procedure similar to that described in the case of E. coli MetRS (MetRS-EC) (Meinnel et al, 1991). Two chro- matographic steps were performed in 10 mM potassium phosphate pH 7.3, 50 mM potassium chloride and 10 mM 2-mercaptoethanol, firstly on a Superose-6 molecular sieve (Pharmacia; 1.6X50 cm; 0.2 ml/min) and secondly on a Q-Hiload anion exchanger (Pharmacia; 1.6X10 cm; 2.5 mumin; 100 mM/h potassium chloride).…”

Section: Methodsmentioning

confidence: 99%

“…2). Mutagenesis studies of E. coli MetRS and selection of second-site mutants showed that the region surrounding Trp461 (called region 460) played a crucia1 role in this process (Ghosh et al, 1990;Meinnel et al, 1991;Schmitt et al, 1993). In addition to Trp461, Asn452 is involved.…”

Section: Characterization Of the Methionine And Zinc Binding Sitesmentioning

confidence: 99%

“…To study the specificities of the above MetRS-BSt variants towards non-methionine tRNAs, the indicator strain PAL121 RAmetCUA was used (Meinnel et al, 1991). This strain carries two indicator genes, lncZ and argE, the translations of each of which are interrupted by an amber stop codon.…”

Section: Characterization Of the Methionine And Zinc Binding Sitesmentioning

confidence: 99%

“…1). However, the most conserved residues are clustered in a limited number of regions, the functional character of which was established in the case of the E. coli enzyme (MetRS-EC; Fourmy et al, 1991Fourmy et al, , 1993bGhosh et al, 1991a,c;Mechulam et al, 1991 a ;Meinnel et al, 1991;Mellot et al, 1989;Schmitt et al, 1993Schmitt et al, , 1994. Consequently, despite a moderate level of amino acid sequence identities, MetRS from various sources may adopt similar 3D structures, with the conserved residues at positions identical to those occurring in the case of the E. coli enzyme.…”

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confidence: 99%

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General Structure/Function Properties of Microbial Methionyl‐Trna Synthetases

Schmitt¹,

Panvert²,

Mechulam³

et al. 1997

European Journal of Biochemistry

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Alignment of the sequences of methionyl-tRNA synthetases from various microbial sources shows low levels of identities. However, sequence identities are clustered in a limited number of sites, most of which contain peptide patterns known to support the activity of the Escherichia coli enzyme. In the present study, site-directed mutagenesis was used to probe the role of these conserved residues in the case of the Bacillus stearothermophilus methionyl-tRNA synthetase. The B. stearothermophilus enzyme was chosen in this study because it can be produced as an active truncated monomeric form, similar to the monomeric derivative of E. coli methionyl-tRNA synthetase produced by mild proteolysis. The two core enzyme molecules share only 27% identical residues. The results allowed the identification of the binding sites for ATP, methionine and tRNA, as well as that responsible for the tight binding of the zinc ion to the enzyme. It is concluded that the thermostable synthetase adopts a three-dimensional folding very similar to that of the E. coli one. Therefore, the two methionyl-tRNA synthetase sequences, although significantly different, maintain a common scafold with the functionally important residues exposed at constant positions. Sequence alignments suggest that the above conclusion can be generalized to the known methionyl-tRNA synthetases from various sources.

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Intron locations and functional deletions in relation to the design and evolution of a subgroup of class I tRNA synthetases

1992

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Selection of suppressor methionyl-tRNA synthetases: mapping the tRNA anticodon binding site.

Abstract: ABSTRACT

Cited by 74 publications

References 27 publications

A study of communication pathways in methionyl- tRNA synthetase by molecular dynamics simulations and structure network analysis

A study of communication pathways in methionyl- tRNA synthetase by molecular dynamics simulations and structure network analysis

General Structure/Function Properties of Microbial Methionyl‐Trna Synthetases

Intron locations and functional deletions in relation to the design and evolution of a subgroup of class I tRNA synthetases

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