Functional Dissection of a Predicted Class-Defining Motif in a Class II tRNA Synthetase of Unknown Structure

Davis, Matthew W.; Buechter, Douglas D.; Schimmel, Paul

doi:10.1021/bi00199a012

Cited by 33 publications

(28 citation statements)

References 34 publications

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“…This may be particularly relevant since tRNA-dependent amino acid recognition was also observed for TrpRS which, in contrast to GlnRS, does not require the presence of tRNA for aminoacyl-adenylate formation. For example, identity elements for both the yeast Asp (e.g., 43) and E. coli Ala (e.g., 44) systems have also been characterized at sub-saturating amino acid concentrations (45,46) and thus might also be susceptible to comparable underestimations of kcat to those described above for tRNAGln variants.…”

Section: Discussionmentioning

confidence: 99%

Interactions between tRNA identity nucleotides and their recognition sites in glutaminyl-tRNA synthetase determine the cognate amino acid affinity of the enzyme.

Ibba

Hong

Sherman

et al. 1996

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

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Sequence-specific interactions between aminoacyl-tRNA synthetases and their cognate tRNAs both ensure accurate RNA recognition and prevent the binding of noncognate substrates. Here we show for Escherichia coli glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1.18) that the accuracy of tRNA recognition also determines the efficiency of cognate amino acid recognition. Steady-state kinetics revealed that interactions between tRNA identity nucleotides and their recognition sites in the enzyme modulate the amino acid affinity of GlnRS. Perturbation of any of the protein-RNA interactions through mutation of either component led to considerable changes in glutamine affinity with the most marked effects seen at the discriminator base, the 10:25 base pair, and the anticodon. Reexamination of the identity set of tRNAGIn in the light of these results indicates that its constituents can be differentiated based upon biochemical function and their contribution to the apparent Gibbs' free energy of tRNA binding. Interactions with the acceptor stem act as strong determinants of tRNA specificity, with the discriminator base positioning the 3' end. The 10:25 base pair and U35 are apparently the major binding sites to GlnRS, with G36 contributing both to binding and recognition. Furthermore, we show that E. coli tryptophanyl-tRNA synthetase also displays tRNA-dependent changes in tryptophan affinity when charging a noncognate tRNA. The ability of tRNA to optimize amino acid recognition reveals a novel mechanism for maintaining translational fidelity and also provides a strong basis for the coevolution of tRNAs and their cognate synthetases.

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Section: Discussionmentioning

confidence: 99%

Interactions between tRNA identity nucleotides and their recognition sites in glutaminyl-tRNA synthetase determine the cognate amino acid affinity of the enzyme.

Ibba

Hong

Sherman

et al. 1996

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

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“…The A76 residues in the first and second models are highlighted in yellow and blue, respectively. Amino acid residues important for the aminoacylation or editing activity (17,28,38,39,45,46) are shown as stick models. Ala-SA in the aminoacylation site and the editing-site zinc ion are depicted as cpk models.…”

Section: Position Of the Editing Domain The Editing Domain Of A Fulmentioning

confidence: 99%

Unique protein architecture of alanyl-tRNA synthetase for aminoacylation, editing, and dimerization

Naganuma

Sekine

Fukunaga

et al. 2009

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

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Alanyl-tRNA synthetase (AlaRS) specifically recognizes the major identity determinant, the G3:U70 base pair, in the acceptor stem of tRNA Ala by both the tRNA-recognition and editing domains. In this study, we solved the crystal structures of 2 halves of Archaeoglobus fulgidus AlaRS: AlaRS-⌬C, comprising the aminoacylation, tRNA-recognition, and editing domains, and AlaRS-C, comprising the dimerization domain. The aminoacylation/tRNA-recognition domains contain an insertion incompatible with the class-specific tRNA-binding mode. The editing domain is fixed tightly via hydrophobic interactions to the aminoacylation/tRNA-recognition domains, on the side opposite from that in threonyl-tRNA synthetase. A groove formed between the aminoacylation/tRNA-recognition domains and the editing domain appears to be an alternative tRNA-binding site, which might be used for the aminoacylation and/or editing reactions. Actually, the amino acid residues required for the G3:U70 recognition are mapped in this groove. The dimerization domain consists of helical and globular subdomains. The helical subdomain mediates dimerization by forming a helixloop-helix zipper. The globular subdomain, which is important for the aminoacylation and editing activities, has a positively-charged face suitable for tRNA binding.crystal structure ͉ dimerization domain ͉ aminoacyl-tRNA synthetase ͉ proofreading ͉ wobble base pair

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“…These lysine residues are also functionally important for stabilization of the ground state and/or the transition state during the formation of aminoacyl-adenylate (36,39,40). In a class II aminoacyl-tRNA synthetase also, a lysine residue that cross-links to the 3Ј-end of periodate-oxidized tRNA is part of a conserved motif (motif 2) important for aminoacyl-adenylate formation and for transfer of the amino acid to the tRNA (41,42). Whether Lys 207 or Lys 210 plays a functional role in MTF is not known.…”

Section: Figmentioning

confidence: 99%

Lysine 207 as the Site of Cross-linking between the 3′-End of Escherichia coli Initiator tRNA and Methionyl-tRNA Formyltransferase

Gite

RajBhandary

1997

Journal of Biological Chemistry

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The specific formylation of initiator methionyl-tRNA by methionyl-tRNA formyltransferase (MTF) is important for initiation of protein synthesis in Escherichia coli. In attempts to identify regions of MTF that come close to the 3-end of the tRNA, we oxidized 32 P-3-endlabeled E. coli initiator methionine tRNA with sodium metaperiodate and cross-linked it to MTF. The crosslinked MTF was separated from uncross-linked MTF by DEAE-cellulose chromatography, and the tRNA in the cross-linked MTF was hydrolyzed with nuclease P1 and RNase T1, leaving behind an oxidized fragment of From assembly and packaging of RNA viruses (1) to mRNA localization during development (2), the specific recognition of RNAs by proteins plays an important role in many biological processes. Examples of these biological processes include RNA processing, RNA splicing, RNA transport, ribosome assembly, translation, and translational regulation (3). As molecules that interact with a variety of different proteins, tRNAs provide an excellent system for studying the molecular basis of specificity in recognition of RNAs by different proteins (4).We are studying the specific recognition of Escherichia coli initiator methionyl-tRNA (Met-tRNA), 1 during its formylation to formylmethionyl-tRNA by the enzyme methionyl-tRNA formyltransferase (MTF, EC 2.1.2.9; 10-formyltetrahydrofolate:L-methionyl-tRNA N-formyltransferase). Formylation of initiator Met-tRNA is important for initiation of protein synthesis in eubacteria and in eukaryotic organelles such as mitochondria and chloroplasts (5-9). In E. coli, formylation provides a positive determinant for allowing the initiation factor IF2 to select the initiator tRNA from other tRNAs (10, 11) and a second negative determinant for blocking the binding of elongation factor EF-Tu to the initiator tRNA (12-14). The formylation reaction is highly specific; the enzyme formylates the initiator Met-tRNA but not the elongator Met-tRNA or any other aminoacyl-tRNA (15). Previous studies have shown that most of the determinants on the initiator tRNA important for its formylation by MTF are clustered in the acceptor stem (16 -19) (Fig. 1). However, although the protein sequence of MTF is known (20), little is known about the amino acid residues in MTF that are important for this recognition and the molecular basis of the specificity in recognition.As a first step in identifying regions of MTF that come close to the acceptor stem of the tRNA, we have cross-linked periodate-oxidized tRNA to MTF and have analyzed the site(s) of cross-linking. We show that Lys 207 in the sequence KLSKE (207-211) of MTF is the site of cross-link to the 3Ј terminus of the tRNA.2 The cross-linking of periodate-oxidized E. coli tRNA fMet to MTF has been studied before by Blanquet and co-workers (21). However, the sites of cross-linking were not analyzed in the previous work. MATERIALS AND METHODSChemicals, Enzymes, and Radioisotopes-Sodium metaperiodate, folinic acid, carboxypeptidase Y (sequencing grade), and methionine were obtained from Sigma. Sod...

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Functional Dissection of a Predicted Class-Defining Motif in a Class II tRNA Synthetase of Unknown Structure

Cited by 33 publications

References 34 publications

Interactions between tRNA identity nucleotides and their recognition sites in glutaminyl-tRNA synthetase determine the cognate amino acid affinity of the enzyme.

Interactions between tRNA identity nucleotides and their recognition sites in glutaminyl-tRNA synthetase determine the cognate amino acid affinity of the enzyme.

Unique protein architecture of alanyl-tRNA synthetase for aminoacylation, editing, and dimerization

Lysine 207 as the Site of Cross-linking between the 3′-End of Escherichia coli Initiator tRNA and Methionyl-tRNA Formyltransferase

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