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 involved in base-specific interactions with the enzyme prevail both in the initial binding recognition and in favouring aminoacylation catalysis. Tertiary nucleotides of base pair G19-C56 and base triple U45-G10-C25 contribute primarily to stabilization of the correctly folded tRNA(Phe) structure, which is important for binding. Other nucleotides of the central core (U20, U16 and of the A26-G44 tertiary base pair) are involved in conformational adjustment of the tRNA upon its interaction with the enzyme. The specificity of nucleotide A73, mutation of which slightly reduces the catalytic rate of aminoacylation, is not displayed at the binding step. A few backbone-mediated contacts of PheRS with the acceptor and anticodon stems revealed in the crystal structure do not contribute to tRNA(Phe) discrimination, their role being limited to stabilization of the complex. The highest affinity of T. thermophilus PheRS for cognate tRNA, observed for synthetase-tRNA complexes, results in 100-3000-fold binding discrimination against non-cognate tRNAs.

Crystal Structure of Human Mitochondrial PheRS Complexed with tRNAPhe in the Active “Open” State

“…The respective tRNA Phe transcripts were synthesized as previously described. 40 Crystallization of the hmPheRS-tRNA Phe complex, data collection, and structure determination…”

Eukaryotic cytosolic and mitochondrial phenylalanyl-tRNA synthetases catalyze the charging of tRNA with the meta -tyrosine

“…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).…”

Structural Basis for Discrimination of L-Phenylalanine from L-Tyrosine by Phenylalanyl-tRNA Synthetase

“…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.…”

“…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.…”