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Query: EC:6.1.1.20 (phenylalanyl-tRNA synthetase)
 358 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The interactions between yeast tRNAphe and phenylalanyl-tRNA synthetase were studied by analysis of the covalent adducts obtained upon monochromatic ultraviolet irradiation at different wavelengths (248, 282, 292, 302 and 313 nm). The high extent of inactivation of phenylalanyl-tRNA synthetase, together with the partial modification of tRNA, as well as the peculiar instability of most of the covalent bonds formed upon irradiation constitute severe limitations to the use of the technique and to the interpretation of the results. These disadvantages led us to select an irradiation wavelength of 248 nm and to use only mild isolation procedures allowing a good recovery of the covalent adducts formed. Seven major tryptic peptides of the enzyme were found to be cross-linked to tRNAPhe whereas six major T1-oligonucleotides were covalently linked to the protein, among these, the three cross-linked oligonucleotides previously described by Shoemaker and Schimmel (J. Biol. Chem. 250 (1975) 4440-4444) in the same system. The difference in the number of covalently linked oligonucleotides is discussed in the light of the instability of the covalent linkages. The localization of the six oligonucleotides at the inside of the two branches forming the L-shaped tRNA molecule is similar to that observed in the yeast valine system (Renaud et al., Eur. J. Biochem. 101 (1979) 475-483) and is consistent with the interaction model previously described (Rich and Schimmel, Nucl. Acids Res. 4 (1977) 1649-1665 and Ebel et al. in Transfer RNA: structure, properties and recognition, (1979) pp. 325-343 Cold Spring Harbor Laboratory, NY). The occurrence of covalent cross-linking upon irradiation in the tryptophan absorption band (302 nm) strongly suggests the participation of this residue in the stabilization of the tRNA enzyme complex.
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PMID:Study of the interaction between yeast tRNAphe and yeast phenylalanyl-tRNA synthetase by monochromatic ultraviolet irradiation at various wavelengths. Advantages and limits of the method. 701 10

The contributions of the natural modified nucleosides to RNA identity in protein/RNA interactions are not understood. We had demonstrated that 15 amino acid long peptides could be selected from a random phage display library using the criterion of binding to a modified, rather than unmodified, anticodon domain of yeast tRNA(Phe) (ASL(Phe)). Affinity and specificity of the selected peptides for the modified ASL(Phe) have been characterized by fluorescence spectroscopy of the peptides' tryptophans. One of the peptides selected, peptide t(F)2, exhibited the highest specificity and most significant affinity for ASL(Phe) modified with 2'-O-methylated cytidine-32 and guanosine-34 (Cm(32) and Gm(34)) and 5-methylated cytidine-40 (m(5)C(40)) (K(d) = 1.3 +/- 0.4 microM) and a doubly modified ASL(Phe)-Gm(34),m(5)C(40) and native yeast tRNA(Phe) (K(d) congruent with 2.3 and 3.8 microM, respectively) in comparison to that for the unmodified ASL(Phe) (K(d) = 70.1 +/- 12.3 microM). Affinity was reduced when a modification altered the ASL loop structure, and binding was negated by modifications that disfavored hairpin formation. Peptide t(F)2's higher affinity for the ASL(Phe)-Cm(32),Gm(34),m(5)C(40) hairpin and fluorescence resonance energy transfer from its tryptophan to the hypermodified wybutosine-37 in the native tRNA(Phe) placed the peptide across the anticodon loop and onto the 3'-side of the stem. Inhibition of purified yeast phenylalanyl-tRNA synthetase (FRS) catalyzed aminoacylation of cognate yeast tRNA(Phe) corroborated the peptide's binding to the anticodon domain. The phage-selected peptide t(F)2 has three of the four amino acids crucial to G(34) recognition by the beta-structure of the anticodon-binding domain of Thermus thermophilus FRS and exhibited circular dichroism spectral properties characteristic of beta-structure. Thus, modifications as simple as methylations contribute identity elements that a selected peptide specifically recognizes in binding synthetic and native tRNA and in inhibiting tRNA aminoacylation.
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PMID:Anticodon domain methylated nucleosides of yeast tRNA(Phe) are significant recognition determinants in the binding of a phage display selected peptide. 1171 72

Introduction of a yeast suppressor tRNA (ytRNA(Phe)(CUA)) and a mutant yeast phenylalanyl-tRNA synthetase (yPheRS (T415G)) into an Escherichia coli expression host allows in vivo incorporation of phenylalanine analogues into recombinant proteins in response to amber stop codons. However, high-fidelity incorporation of non-natural amino acids is precluded in this system by mischarging of ytRNA(Phe)(CUA) with tryptophan (Trp) and lysine (Lys). Here we show that ytRNA(Phe)(CUA) and yPheRS can be redesigned to achieve high-fidelity amber codon suppression through delivery of p-bromophenylalanine (pBrF). Two strategies were used to reduce misincorporation of Trp and Lys. First, Lys misincorporation was eliminated by disruption of a Watson-Crick base pair between nucleotides 30 and 40 in ytRNA(Phe)(CUA). Loss of this base pair reduces mischarging by the E. coli lysyl-tRNA synthetase. Second, the binding site of yPheRS was redesigned to enhance specificity for pBrF. Specifically, we used the T415A variant, which exhibits 5-fold higher activity toward pBrF as compared to Trp in ATP-PP(i) exchange assays. Combining mutant ytRNA(Phe)(CUA) and yPheRS (T415A) allowed incorporation of pBrF into murine dihydrofolate reductase in response to an amber codon with at least 98% fidelity.
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PMID:Design of a bacterial host for site-specific incorporation of p-bromophenylalanine into recombinant proteins. 1695 16

A designed yeast phenylalanyl-tRNA synthetase (yPheRS (T415G)) activates four tryptophan (Trp) analogues (6-chlorotryptophan (6ClW), 6-bromotryptophan (6BrW), 5-bromotryptophan (5BrW), and benzothienylalanine (BT)) that are not utilized by the endogenous E. coli translational apparatus. Introduction of yPheRS (T415G) and a mutant yeast phenylalanine amber suppressor tRNA (ytRNAPheCUA_UG) into an E. coli expression host allowed site-specific incorporation of three of these analogues (6ClW, 6BrW, and BT) into recombinant murine dihydrofolate reductase in response to amber stop codons with at least 98% fidelity. All three analogues were introduced at the Trp66 position in the chromophore of a cyan fluorescent protein variant (CFP6) to investigate the attendant changes in spectral properties. Each of the analogues caused blue shifts in the fluorescence emission and absorption maxima. The CFP6 variant bearing BT at position 66 exhibited an unusually large Stokes shift (56 nm). An expanded set of genetically encoded Trp analogues should enable the design of new proteins with novel spectral properties.
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PMID:Site-specific incorporation of tryptophan analogues into recombinant proteins in bacterial cells. 1768 15

A yeast phenylalanyl-tRNA synthetase variant with T415G mutation (yPheRS (T415G)) was rationally designed to recognize various phenylalanine (Phe) analogs allowing site-specific incorporation into an amber site of a protein in E. coli. However, the relaxed substrate specificity of yPheRS (T415G) led to a significant tryptophan (Trp) misincorporation restricting the utility of yPheRS for biosynthesis of proteins containing a Phe analog. In order to obtain yPheRS variants with high substrate-specificity toward a Phe analog, we developed a general high-throughput screening method. This method uses fluorescence reduction of green fluorescence protein (GFP) upon efficient introduction of a Phe analog into multiple sites of GFP by breaking the degeneracy of the Phe codons. Combined use of positive and negative screenings of a yPheRS saturation library led to a yPheRS variant (yPheRS_naph) very selective toward 2-l-naphthylalanine (2Nal), a model Phe analog. The yPheRS_naph exhibited 6-fold higher relative activity toward 2Nal (vs Trp) in ATP-PPi exchange assays and led to high-fidelity incorporation of 2Nal into an amber site of murine dihydrofolate reductase in both minimal and rich media. These results successfully demonstrate that the high-throughput screening method developed can be used to evolve yPheRS to be very selective toward a Phe analog.
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PMID:Tailoring the substrate specificity of yeast phenylalanyl-tRNA synthetase toward a phenylalanine analog using multiple-site-specific incorporation. 2526 49