Bacterial tyrosyl-tRNA synthetases (TyrRS) possess ā exibly linked C-terminal domain of~80 residues, which has hitherto been disordered in crystal structures of the enzyme. We have determined the structure of Thermus thermophilus TyrRS at 2.0 A Ê resolution in a crystal form in which the C-terminal domain is ordered, and con®rm that the fold is similar to part of the C-terminal domain of ribosomal protein S4. We have also determined the structure at 2.9 A Ê resolution of the complex of T.thermophilus TyrRS with cognate tRNA tyr (GYA). In this structure, the C-terminal domain binds between the characteristic long variable arm of the tRNA and the anti-codon stem, thus recognizing the unique shape of the tRNA. The anticodon bases have a novel conformation with A-36 stacked on G-34, and both G-34 and Y-35 are base-speci®cally recognized. The tRNA binds across the two subunits of the dimeric enzyme and, remarkably, the mode of recognition of the class I TyrRS for its cognate tRNA resembles that of a class II synthetase in being from the major groove side of the acceptor stem. Keywords: class I aminoacyl-tRNA synthetase/ribosomal protein S4/tRNA recognition/tyrosyl-tRNA synthetase/ X-ray crystallography IntroductionTyrosyl-tRNA synthetase (TyrRS) is a class I aminoacyltRNA synthetase, but is unusual in that it is a functional dimer, a feature only shared with tryptophanyl-tRNA synthetase amongst class I synthetases (Cusack, 1995). It was the ®rst synthetase to have its crystal structure solved (Bhat et al., 1982), including its substrate complexes with tyrosine and tyrosyl-adenylate (Brick and Blow, 1987;Brick et al., 1989), and both the mechanism of tyrosyladenylate formation (reviewed in Fersht, 1987;First, 1997) and its interaction with cognate tRNA tyr (reviewed in Bedouelle, 1990;Bedouelle et al., 1993) have been the subject of intense biochemical study. However, there remain a number of signi®cant areas where structural data that correlate with biochemical observations are lacking. Here we report the ®rst crystal structure of a bacterial tyrosyl-tRNA synthetase complexed with cognate tRNA tyr . This permits us to visualize the mode of interaction of this synthetase for its cognate tRNA, which in prokaryotes, but not archaea or eukaryotes, is of the class 2 type, i.e. with a long variable arm. The enzyme subunit comprises an N-terminal Rossmann-fold catalytic domain, characteristic of class I synthetases, followed by a central a-helical domain and, ®nally, a putative tRNAbinding C-terminal domain of~80 residues, which has been invisible until now due to disorder in all crystal structures of TyrRS (Brick et al., 1989;Qiu et al., 2001). The structure described here reveals the exact role in speci®c tRNA recognition of the¯exibly linked C-terminal domain and also shows how unique tertiary interactions in the core of tRNA tyr lead to a different orientation of the long variable arm, permitting discrimination from other long variable arm (class 2) tRNAs such as tRNA ser . The structure con®rms the expected cross-subun...

The complex between Thermus thermophilus prolyl-tRNA synthetase (ProRSTT) and its cognate tRNA has been crystallized using two different isoacceptors of tRNA(Pro). Similar bipyramidal crystals of the complexes of ProRSTT with the two different tRNA(Pro) isoacceptors grow within two weeks from 32% saturated ammonium sulfate solution. They belong to space group P4(3)2(1)2, with unit-cell parameters a = 143.1, b = 143.1, c = 228.6 A. The crystals diffract weakly to a maximum resolution of 3.1 A. Superior quality crystals were obtained by growing slowly from precipitate over 5-6 months. These are of the same space group but have slightly altered unit-cell parameters, a = 140.8, b = 140.8, c = 237.0 A. These crystals diffract more strongly to at least 2.8 A resolution and a complete data set to 2.85 A resolution has been collected from a single crystal. Comparison of the packing in the two crystal forms shows that domain flexibility contributes to the presence of different crystal contacts in the two forms.

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The maintenance of amino acid specificity by aminoacyl-tRNA synthetases can require the hydrolysis of missynthesized products that is known as amino acid editing. Bacterial prolyl-tRNA synthetase includes a special editing domain, that deacylates alanyl-tRNAPro, and so exhibits post-transfer editing activity. The mechanism of tRNA-dependent editing by prolyl-tRNA synthetase has to be defined. The present work aim is to study the structure of the active site of enterobacteria E. faecalis prolyl-tRNA synthetase editing domain. The amino acids positions E218, T257, K279, G331, S332, G334, and H366 have been chosen for the site-directed mutagenesis (alanine scanning). An editing activity of the mutants was compared with the wild type prolyl-tRNA synthetase. Three amino acid residues, important for the editing activity, K279, G331 and H366, were revealed. This data are consistent with the existing suppositions about the structure of bacterial prolyl-tRNA synthetase deacylating active sit

Aminoacyl tRNA synthetases are enzymes that specifically attach amino acids to cognate tRNAs for use in the ribosomal stage of translation. For many aminoacyl tRNA synthetases, the required level of amino acid specificity is achieved either by specific hydrolysis of misactivated aminoacyl-adenylate intermediate (pre-transfer editing) or by hydrolysis of the mischarged aminoacyl-tRNA (post-transfer editing). To investigate the mechanism of post-transfer editing of alanine by prolyl-tRNA synthetase from the pathogenic bacteria Enterococcus faecalis, we used molecular modeling, molecular dynamic simulations, quantum mechanical (QM) calculations, site-directed mutagenesis of the enzyme, and tRNA modification. The results support a new tRNA-assisted mechanism of hydrolysis of misacylated Ala-tRNA. The most important functional element of this catalytic mechanism is the 2'-OH group of the terminal adenosine 76 of Ala-tRNA, which forms an intramolecular hydrogen bond with the carbonyl group of the alanine residue, strongly facilitating hydrolysis. Hydrolysis was shown by QM methods to proceed via a general acid-base catalysis mechanism involving two functionally distinct water molecules. The transition state of the reaction was identified. Amino acid residues of the editing active site participate in the coordination of substrate and both attacking and assisting water molecules, performing the proton transfer to the 3'-O atom of A76.

In prokaryotic cells three tRNA species, tRNASer, tRNALeu and tRNATyr, possess a long variable arm of 11–20 nucleotides (type 2 tRNA) rather than usual 4 or 5 nucleotides (type 1 tRNA). In this review we have summarized the results of our research on the structural basis for recognition and discrimination of type 2 tRNAs by Thermus thermophilus seryl-, tyrosyl- and leucyl-tRNA synthetases (SerRS, TyrRS and LeuRS) obtained by X-ray crystallography and chemical probing tRNA in solution. Crystal structures are now known of all three aminoacyl-tRNA synthetases complexed with type 2 tRNAs and the different modes of tRNA recognition represented by these structures will be discussed. In particular, emphasis will be given to the results on recognition of characteristic shape of type 2 tRNAs by cognate synthetases. In tRNASer, tRNATyr and tRNALeu the orientation of the long variable arm with respect to the body of the tRNA is different and is controlled by different packing of the core. In the case of SerRS the N-terminal domain and in the case of TyrRS, the C-terminal domain, bind to the characteristic long variable arm of the cognate RNA, thus recognizing the unique shape of the tRNA. The core of T. thermophilus tRNALeu has several layers of unusual base-pairs, which are revealed by the crystal structure of tRNALeu complexed with T. thermophilus LeuRS and by probing a ligand-free tRNA by specific chemical reagents in solution. In the crystal structure of the LeuRS-tRNALeu complex the unique D-stem structure is recognized by the C-terminal domain of LeuRS and these data are in good agreement with those obtained in solution. LeuRS has canonical class I mode of tRNA recognition, approaching the tRNA acceptor stem from the D-stem and minor groove of the acceptor stem side. SerRS also has canonical class II mode of tRNA recognition and approaches tRNASer from opposite, variable stem and major groove of acceptor stem site. And finally, TyrRS in strong contrast to canonical class I system has class II mode of tRNA recognition

Методами хімічної модифікації вивчено реакційну здатність азотистих основ, шр входять до складу тРНК 1 *" із Т. thermophilus. Одержані результати свідчать про існування молекул тРНК 1 *" в розчині у вигляді канонічної L-форми. В корі молекули виникають триплетні взаємодії (8-14)-21у (13-22J-9 і (15-48)~20а. Дві останні є характерною особливістю відомих просторо вих структур тРНК 11 класу. Встановлено, шр залишки 15 і 21 мають різну реакційну здатність у mPHK heu і тРНК* ег із Т. thermophilus, шр вказує на відмінності у просторовій будові корових частин цих тРНК.

Інститут молекулярної біології і генетики ИЛИ України Вул. Академіка Заболотного, 150, Киї», 03143, Україна Методами хімічної модифікації вивчено реакційну здатність залишків фосфорної кислоти та азотистих основ, що входять до складу mPHJC* 1 ' з Т. thermophilus. Отримані результати свідчать про надзвичайну близкість просторової структури inPIffC Str у розчині до такої в кристалі комплексу тРНК 3 * 1 Т. thermophilus з серші-тРНК синтета:юю.