2002
DOI: 10.1002/jmr.575
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tRNA discrimination by T. thermophilus phenylalanyl–tRNA synthetase at the binding step

Abstract: The extent of tRNA recognition at the level of binding by Thermus thermophilus phenylalanyl-tRNA synthetase (PheRS), one of the most complex class II synthetases, has been studied by independent measurements of the enzyme association with wild-type and mutant tRNA(Phe)s as well as with non-cognate tRNAs. The data obtained, combined with kinetic data on aminoacylation, clearly show that PheRS exhibits more tRNA selectivity at the level of binding than at the level of catalysis. The anticodon nucleotides involve… Show more

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Cited by 13 publications
(15 citation statements)
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“…The respective tRNA Phe transcripts were synthesized as previously described. 40 Crystallization of the hmPheRS-tRNA Phe complex, data collection, and structure determination…”
Section: Methodsmentioning
confidence: 99%
“…The respective tRNA Phe transcripts were synthesized as previously described. 40 Crystallization of the hmPheRS-tRNA Phe complex, data collection, and structure determination…”
Section: Methodsmentioning
confidence: 99%
“…PheRSs from natural or overexpressed sources were purified as described (23)(24)(25). The tRNA Phe s were synthesized by using runoff transcription of synthetic genes with T7 RNA polymerase followed by electrophoretic isolation of the correct-length transcripts as described (26). To prepare labeled tRNAs, transcriptions were run in the presence of ␣-[ 32 P]ATP (Amersham Biosciences).…”
Section: Methodsmentioning
confidence: 99%
“…PheRS and native tRNA Phe (isoacceptor I, 1700 pmol of Phe/A 260 unit) from T. thermophilus HB8 were isolated and purified as described (Ankilova et al, 1988;Stepanov et al, 1998). E. coli tRNA Phe was synthesized by using runoff transcription of a synthetic gene with T7 RNA polymerase, followed by electrophoretic isolation of the correct length transcript as described (Vasil'eva et al, 2002). Plasmid DNA containing a gene of E. coli tRNA Phe under control of the phage T7 promoter was a gift from O.C.…”
Section: Materials and General Methodsmentioning
confidence: 99%
“…tRNA Aminoacylation Aminoacylation of tRNA Phe (native T. thermophilus or E. coli tRNA Phe transcript) with T. thermophilus PheRS was performed in conditions described previously (Vasil'eva et al, 2002). Direct attachment of noncognate amino acids to tRNA Phe was measured in similar conditions by the addition of 100 mM L-[ 14 C]Tyr, L-[ 14 C]Ile, or L-[ 14 C]His or 20 mM L-[ 3 H]Trp to the reaction mixture instead of 14 C/ 3 H-labeled Phe.…”
Section: Materials and General Methodsmentioning
confidence: 99%