Publications (35) View all
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Article: A domain in the N-terminal extension of class IIb eukaryotic aminoacyl-tRNA synthetases is important for tRNA binding.
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ABSTRACT: Cytoplasmic aspartyl-tRNA synthetase (AspRS) from Saccharomyces cerevisiae is a homodimer of 64 kDa subunits. Previous studies have emphasized the high sensitivity of the N-terminal region to proteolytic cleavage, leading to truncated species that have lost the first 20-70 residues but that retain enzymatic activity and dimeric structure. In this work, we demonstrate that the N-terminal extension in yeast AspRS participates in tRNA binding and we generalize this finding to eukaryotic class IIb aminoacyl-tRNA synthetases. By gel retardation studies and footprinting experiments on yeast tRNA(Asp), we show that the extension, connected to the anticodon-binding module of the synthetase, contacts tRNA on the minor groove side of its anticodon stem. Sequence comparison of eukaryotic class IIb synthetases identifies a lysine-rich 11 residue sequence ((29)LSKKALKKLQK(39) in yeast AspRS with the consensus xSKxxLKKxxK in class IIb synthetases) that is important for this binding. Direct proof of the role of this sequence comes from a mutagenesis analysis and from binding studies using the isolated peptide.The EMBO Journal 06/2000; 19(10):2371-80. · 9.20 Impact Factor -
Article: tRNA mimics.
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ABSTRACT: Mimics recapitulating the structural features of tRNAs are involved in biological processes other than ribosome-dependent protein synthesis. A knowledge of the rules underlying the architecture and function of tRNAs allows the design of non-natural mimics. The study of these mimics sheds light upon links between replication, translation and metabolic pathways, leads to biotechnological applications, and provides experimental and conceptual tools for the exploration of primordial evolutionary processes.Current Opinion in Structural Biology 07/1998; 8(3):286-93. · 9.42 Impact Factor -
Article: Sequences outside recognition sets are not neutral for tRNA aminoacylation. Evidence for nonpermissive combinations of nucleotides in the acceptor stem of yeast tRNAPhe.
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ABSTRACT: Phenylalanine identity of yeast tRNAPhe is governed by five nucleotides including residues A73, G20, and the three anticodon nucleotides (Sampson et al., 1989, Science 243, 1363-1366). Analysis of in vitro transcripts derived from yeast tRNAPhe and Escherichia coli tRNAAla bearing these recognition elements shows that phenylalanyl-tRNA synthetase is sensitive to additional nucleotides within the acceptor stem. Insertion of G2-C71 has dramatic negative effects in both tRNA frameworks. These effects become compensated by a second-site mutation, the insertion of the wobble G3-U70 pair, which by itself has no effect on phenylalanylation. From a mechanistic point of view, the G2-C71/G3-U70 combination is not a "classical" recognition element since its antideterminant effect is compensated for by a second-site mutation. This enlarges our understanding of tRNA identity that appears not only to be the outcome of a combination of positive and negative signals forming the so-called recognition/identity set but that is also based on the presence of nonrandom combinations of sequences elsewhere in tRNA. These sequences, we name "permissive elements," are retained by evolution so that they do not hinder aminoacylation. Likely, no nucleotide within a tRNA is of random nature but has been selected so that a tRNA can fulfill all its functions efficiently.Journal of Biological Chemistry 06/1998; 273(19):11605-10. · 4.77 Impact Factor -
Article: Subtle atomic group discrimination in the RNA minor groove.
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ABSTRACT: As a problem in molecular recognition and for drug discovery, great interest has developed around the possibility that RNA structures could be discriminated by peptides and other small molecules. Although small peptides have been shown to have the capacity to discriminate specific bulges and loops in RNA molecules, discrimination of double helical regions by a peptide binder has not been reported. Indeed, the most accessible part of an RNA helix is the minor groove, and fundamental stereochemical considerations have suggested that discrimination of at least some base pairs would be difficult in the minor groove. Here we report the design and isolation of a peptide binder that manifests the most subtle kind of discrimination of base pair differences in the RNA minor groove. Functional discrimination of a single atomic group is demonstrated as well as the difference between two different angular orientations of the same group. This report of RNA helix discrimination by a peptide binder suggests a richer potential for RNA minor groove recognition than previously thought.Proceedings of the National Academy of Sciences 11/1997; 94(21):11291-4. · 9.68 Impact Factor -
Article: Peptides for RNA discrimination and for assembly of enzymes that act on RNA.
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ABSTRACT: RNA recognition by tRNA synthetases is thought to have arisen by the recruitment of RNA binding peptide elements into the frameworks of primordial enzymes that carried out amino acid activation. While peptides have been shown to have the capacity to discriminate irregular RNA structures such as bulges and loops, the sequence-specific recognition of base pairs in RNA helices like those in tRNAs had not been demonstrated. Such discrimination by peptide binders was thought to be inherently difficult. But in this work we show that discrimination of a single base pair in an RNA helix may be achieved with a rationally designed, chemically synthesized peptide. In a separate study, we demonstrated the noncovalent association of a peptide binder with an amino acid activation domain to give an active and specific tRNA synthetase. Thus, peptides with high specificity for an RNA helix can be obtained. In addition, a peptide can associate with another protein to give an enzyme that acts on RNA. The principles suggested by these studies may be general.Nucleic Acids Symposium Series 02/1997;
