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.

The small-angle X-ray scattering technique (SAXS) is proposed for the investigation of equilibrium macromolecular interactions of the enzyme-substrate type in solution. Experimental procedures and methods of analysing the data obtained from SAXS have been elaborated. The algorithm for the data analysis allows one to determine the stoichiometric, equilibrium and structural parameters of the enzyme-substrate complexes obtained. The thermodynamic characteristics for the formation of complexes of tRNAphe with phenylalanyl-tRNA synthetase have been determined and demonstrate negative cooperativity for binding of the two tRNAPhe molecules. The structural parameters (I$, &, semi-axes) have been determined for free phenylalanyl-tRNA synthetase and tRNAPhe from E. coli MRE-600 and of enzyme complexes possessing one and two tRNAPhe molecules, indicating structural rearrangements of the enzyme in the interaction with tRNAPhe.Small-angle X-ray scattering; Phenylalanyl-tRNA synthetase; Enzyme-substrate interaction; tRNAPhe

Phenylalanyl-tRNA synthetase from Thermus thermophilus HB8 was isolated, characterized and crystallized. The enzyme is a tetramer of a&-type structure, its molecular mass being 264 kDa. (Thermus thermophilus)

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.

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