The mode of interactions with tRNA explains the absolute necessity for the (alphabeta)2 architecture of PheRS. The interactions of tRNAPhe with PheRS and particularly with the coiled-coil domain of the alpha subunit result in conformational changes in TPsiC and D loops seen by comparison with uncomplexed yeast tRNAPhe. The tRNAPhe is a newly recognized type of RNA molecule specifically interacting with the RBD fold. In addition, a new type of anticodon-binding domain emerges in the aaRS family. The uniqueness of PheRS in charging 2'OH of tRNA is dictated by the size of its adenine-binding pocket and by the local conformation of the tRNA's CCA end.

The crystal structure of phenylalanyl-tRNA synthetase (PheRS) from Thermus thermophilus, a class II aminoacyl-tRNA synthetase, complexed with phenylalanyl-adenylate (Phe-AMP) was determined at 2.6 A resolution. Crystals of native PheRS were soaked in a solution containing phenylalanine and ATP in the presence of Mn(2+) ions. The first step of the aminoacylation reaction proceeds within the crystals, resulting in Phe-AMP formation at the active site. Specific recognition of the phenylalanine portion of the Phe-AMP is achieved by interactions of the phenyl ring of Phe-AMP with two neighbouring residues, Phealpha258 and Phealpha260. No manganese ions were observed within the active site; their role in the formation of the transition state may be assigned to a number of polar residues and water molecules. In the anomalous Fourier difference map, a divalent metal ion was detected at the interface of the alpha- and beta-subunits at a short distance from motif 3 residues participating in the substrate binding. A sulfate ion, which was identified on the protein surface, may mediate the interactions of PheRS with DNA. Visible conformational changes were detected in the active-site area adjacent to the position of the Phe-AMP, compared with the structure of PheRS complexed with a synthetic adenylate analogue (phenylalaninyl-adenylate). Based on the known structures of the substrate-free enzyme and its complexes with various ligands, a general scheme for the phenylalanylation mechanism is proposed.

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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.