Identification of the tRNA anticodon recognition site of Escherichia coli methionyl-tRNA synthetase

Abstract

We have previously shown that the anticodon of methionine tRNAs contains most, if not all, of the nucleotides required for specific recognition of tRNA substrates by Escherichia coli methionyl-tRNA synthetase [Schulman, L. H., & Pelka, H. (1988) Science 242, 765-768]. Previous cross-linking experiments have also identified a site in the synthetase that lies within 14 A of the anticodon binding domain [Leon, O., & Schulman, L. H. (1987) Biochemistry 26, 5416-5422]. In the present work, we have carried out site-directed mutagenesis of this domain, creating conservative amino acid changes at residues that contain side chains having potential hydrogen-bond donors or acceptors. Only one of these changes, converting Trp461----Phe, had a significant effect on aminoacylation. The mutant enzyme showed an approximately 60-100-fold increase in Km for methionine tRNAs, with little or no change in the Km for methionine or ATP or in the maximal velocity of the aminoacylation reaction. Conversion of the adjacent Pro460 to Leu resulted in a smaller increase in Km for tRNA(Mets), with no change in the other kinetic parameters. Examination of the interaction of the mutant enzymes with a series of tRNA(Met) derivatives containing base substitutions in the anticodon revealed sequence-specific interactions between the Phe461 mutant and different anticodons. Km values were highest for tRNA(mMet) derivatives containing the normal anticodon wobble base C. Base substitutions at this site decreased the Km for aminoacylation by the Phe461 mutant, while increasing the Km for the wild-type enzyme and for the Leu460 mutant to values greater than 100 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural similarities in glutaminyl- and methionyl-tRNA synthetases suggest a common overall orientation of tRNA binding.

“…This conserved motif at the inside corner of the tRNA is highlighted in black. Lysine and tryptophan residues of Met-tRNA synthetase that have been shown by biochemical means (19,21,24) to be in proximity to the anticodon and D loop are indicated by black dots. Two loops (closer loop residues 261-267, farther loop 235-237) of the Met-tRNA synthetase enzyme project into the major groove near the junction of the acceptor and T stems.…”

“…Chemical cross-linking studies suggest that Lys-465 on this helix is within 14 A of the anticodon of the tRNA (21), and site-directed mutagenesis data have further implied a role in binding for residue Trp461 (19,20). Lys-402 and Lys439, located as well in the carboxyl-terminal helical domain, also have been implicated by chemical cross-linking (24).…”

“…In the case of the Met-tRNA synthetase-tRNAMet interaction, chemical cross-linking studies have identified enzyme residues Lys-61 and Lys-335 as being adjacent to the 3' end of the bound tRNA (17), and site-directed mutagenesis has demonstrated that Lys-335 plays a crucial role in amino acid activation (18). Cross-linking and site-directed mutagenesis studies have also been used to identify residues Trp-461 and Lys-465 as being located near the anticodon of the tRNA (19)(20)(21). These data thus establish two widely separated regions of each macromolecule that lie in close proximity to the structural interface that forms upon binding.…”

In vivo selection of lethal mutations reveals two functional domains in arginyl–tRNA synthetase

“…The crystallographic structure of class I yeast ArgRS has recently been determined at 2+75Å resolution in the presence of arginine (Cavarelli et al+, 1998) (Brunie et al+, 1990;Ghosh et al+, 1990Ghosh et al+, , 1991Meinnel et al+, 1991;Kim et al+, 1993b;Schmitt et al+, 1993)+ The center of this second cluster is ;30 Å from the arginine molecule present in the active site+ Some of the selected mutations affect the class I signature sequences+ In the HIGH-like sequence (HA 160 G 161 H), the following two mutations were found: AG160 and GD161+ Natural variations are observed in ArgRS sequences at position 160 (Ala, Val, Leu, Ile, and Met, but no Gly) whereas residue Gly161 is strictly conserved in all sequenced ArgRSs+ In the KMSKS-like sequence (GMS 409 TR 411 sequence in the yeast enzyme) we isolated the following two mutations: SL409 and RT411+ Residue 411 is a strictly conserved Arg, whereas at position 409, half of the ArgRS sequences contain a Lys residue (an Arg residue is also observed in Arabidopsis thaliana ArgRS)+ Other lethal mutations affecting invariant residues are EK294, DH351, DN351, YH491, and RM495+ Seven lethal mutations (SF165, GD190, GD197, TI349, GC403, GD403, GN403, and GS483) concern residues conserved in a subclass of ArgRS containing the eukaryotic enzymes as well as some bacterial enzymes (E. coli, Yersinia pestis, Enterobacter faecalis, Neisseria meningococus, Neisseria gonorrhoeae, Haemophilus influenzae, Pseudomonas aeruginosa)+ Eight mutations affect residues presenting important natural variations (GD202, RK350, AT372, AV372, PL446, AV450, SL494, and HY559)+ Here we want to mention that a Lys350 and a Leu446 are naturally found in some species+…”

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

“…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). Two negatively charged residues, Asp-449 and Asp-456, also contribute to the overall specificity of the anticodon recognition, although they do not make direct contact with tRNA (11).…”