Recognition of tRNA^Phe by Phenylalanyl‐tRNA Synthetase of Thermus Thermophilus

Abstract

The tRNAYh' nucleotides required for recognition by phenylalanyl-tRNA synlhetase of lhermus thermophilus have been determined using Escherichia roli tRNAph' transcripts with various mutations. The anticodon nucleotides are shown to be the most important recognition elements. The discriininator nucleotide, A73, involved in thc recognition sel of yeast, E. r d i and human phenylalanyl-tRNA synthetases contributes only slightly to tRNAPh' recognition by Th. tht,rrrmphilus phenylaliinyl-tRNA synthetase. Nucleotide 20 and some tertiary nucleotides, including the conserved GI 9 . CS6 base pair, are proposed to participate in stabilization of the precise tRNA conformation required for efficient aminoxcylation. The role of the 3'-CCA terminus, corninon to all tRNAs, in the spccific intcraction of tRNA with phenylalanyltRNA synthetase is discussed.Keyworh: tRNA; aminoacyl-tRNA synthetase; 1'7 transcript; IKNA recognition.Although all aminoacyl-tRNA synthctases have a coinrnon function in protein biosynthesis, namely to catalyze the aminoacylation of tRNA with one specific cognate amino acid, they are widely diverse in structure. All phenylalanyl-tRNA synihetases (tRNA'"" synthetases) known have a rare (for aminoacyltRNA synthetases) subunit structure of the (I$, type [I], excepting mitochondria1 tRNAPhc synthetase from Succ:harnmyces cerevisiae 121. Recent X-ray crystallographic analysis of a number of aminoacyl-tRNA synthetases and computer comparison of amino acid sequences have revealed two groups of aininoacyl-tRNA synthetases [31. tKNA"hr synthetases belong to the second class of aminoacyl-tRNA synthetases. For all enzymes of this class, tRNA aminoacylation occurs on the 3'-hydroxyl group of the 3'-terminal adenosine residue. However, tRNAphe synthetase iicylates tRNAYh' on the 2'-hydroxyl group [4, 51. tRNAPh' synthetase of Thermus thermophilus can attach the phenylalanyl moiety to both 2'-OH and 3'-OH groups simultaneously 161, tRNAPhe synthetase of Th. thermoyhilus is the biggest aminoacyl-tRNA synthetase crystallized to data. A high-resolution Xray study o f this enzyme and its cocrystals with tRNAPhcis now in progress [7]. tRNA'"* synthetase i s one enzyme for which the tRNA recognition set is known for evolutionary diverged species. The recognition patterns for yeast [S] and human [9] tRNAph' synthetases seem to be very similar and include three anticodon nucleotides (G34, A35 and A36), G20 and A73. The only difference between them is the importance of one or two anticodon stem base pairs for the recognition of tRNAPhr by the human enzyme.

tRNA discrimination by T. thermophilus phenylalanyl–tRNA synthetase at the binding step

“…The mutants (Moor et al, 1995). Nucleotides whose mutations appeared to have a very modest, if any, effect are among the contact sites in the crystal structure of the complex (Goldgur et al, 1997) or were supposed to be involved in conformational rearrangement of the complex .…”

“…Closely similar kinetic parameters of aminoacylation with T. thermophilus PheRS have been determined for the native T. thermophilus tRNA Phe and the E. coli tRNA Phe transcript at two MgCl 2 concentrations (9 and 15 mM); 9 mM MgCl 2 is optimal for efficient aminoacylation of both tRNAs. Mutant transcripts obtained on the background of E. coli tRNA Phe were further used to identify the tRNA nucleotides recognized by the thermophilic PheRS (Moor et al, 1995).…”

“…The (ab) 2 subunit structure of cytoplasmic PheRS is markedly conserved through evolution (for a review see Meinnel et al, 1995). The tRNA Phe nucleotides required for recognition by PheRSs of evolutionarily diverged species have been analysed by in vitro studies (Sampson et al, 1990Nazarenko et al, 1992;Perret et al, 1992;Tinkle Peterson and Uhlenbeck, 1992;Moor et al, 1992Moor et al, , 1995Aphasizhev et al, 1996). The structure of the complex of T. thermophilus PheRS with cognate tRNA Phe has been extensively investigated by X-ray analysis (Goldgur et al, 1997) and biochemical approaches (Kreutzer et al, 1995;Moor et al, 2001).…”

An Example of Non‐Conservation of Oligomeric Structure in Prokaryotic Aminoacyl‐tRNA Synthetases

“…Therefore, this system resembles various mesophilic and thermophilic systems in which post-transcriptional modifications do not participate in tRNA identity (e.g. Piitz et al, 1991 ;Nameki et al, 1992;Moor et al, 1995;Becker et al, 1996). Structural peculiarities and specificity for prokaryotic and eukaryotic tRNAG'' species.…”

Cross-species aminoacylation of tRNA with a long variable arm between Escherichia coli and Saccharomyces cerevisiae

“…Recent studies have shown that identity elements of a tRNA often vary during evolution (9)(10)(11)(12)(13)(14)(15)(16), although some are maintained (17)(18)(19)(20). An artificial aminoacylation system comprised of a tRNA and an aminoacyl-tRNA synthetase from different sources often causes a unilateral aminoacylation specificity and sometimes causes a lack of amino acid specificity (21)(22)(23).…”