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PubMedDate not supplied

[Proteins contacting with peptidyl-tRNA at the A-site of the Escherichia coli ribosome after enzymatic and non-enzymatic binding of aminoacyl-tRNA].

Proteins contacting (directly interacting) with peptidyl-tRNA in the A-site of E. coli ribosome were determined by means of ultraviolet-induced RNA-protein cross-linking. It has been shown that upon enzymatic binding of Phe-tRNAPhe with the posttranslocated ribosome and following transpeptidation, the peptidyl-tRNAPhe directly interacts with proteins S5, S10, L6, L16 and S13/S14/L27, while upon non-enzymatic binding--with S5, S10, L2, L6 and L16. These data evidenced, that the difference in tRNA-protein interactions upon enzymatic and non-enzymatic binding of Phe-tRNAPhe to the ribosome does not prevent the following step and remains after transpeptidation.

Escherichia coli↗
PubMed1994

[Nucleotide sequence and organization of tRNA-Gly (UCC), tRNA-Arg (UCU), and alpha-subunits of CF1ATPase in chloroplast DNA of Allium porrum].

The nucleotide sequence of 1029 bp BamHI-fragment of leek chloroplast DNA (Allium porrum, fam. Liliaceae) has been determined. The fragment contains the 3'-terminal part of the tRNA-Gly (UCC) gene, the tRNA-Arg (UCU) gene and the 3'-terminal domain of the CF1ATPase alpha-subunit gene. The gene arrangement and the nucleotide sequence of this fragment are similar to those of the tobacco chloroplast DNA but differ significantly from that of other monocots in which the region containing these genes underwent extensive recombination.

Allium↗
PubMed1984

A Chinese hamster ovary leucyl-tRNA synthetase mutant with a uniquely altered high molecular weight leucyl-tRNA synthetase complex.

The Chinese hamster ovary (CHO) cell culture temperature-sensitive mutant ts025Cl with a defect in leucyl-tRNA synthetase ( LeuRS ) does not have an inherently more thermolabile LeuRS , but instead the mutation causes the complete loss of the LeuRS high molecular weight complexes which are present in normal wild-type cells. The mutant cell LeuRS has a single 8 S enzyme form which corresponds hydrodynamically to the 8 S free form of wild-type enzyme. Both 8 S forms have the same thermostability and the same Km for leucine, indicating that there is no inherent defect in the catalytic activity of the enzyme. The temperature-sensitive phenotype can be explained by the lack of thermostable high molecular weight forms of LeuRS .

Amino Acyl-tRNA Synthetases↗
PubMed1989

Evidence that the 3' end of a tRNA binds to a site in the adenylate synthesis domain of an aminoacyl-tRNA synthetase.

Aminoacylation requires that an enzyme-bound aminoacyladenylate is brought proximal to the 3' end of a specific transfer RNA. In Escherichia coli alanyl-tRNA synthetase, the first 368 amino acids encode a domain for adenylate synthesis while sequences on the carboxyl-terminal side of this domain are required for much of the enzyme-tRNAAla binding energy. The 3' end of E. coli tRNAAla has been cross-linked to the enzyme, and sequence analysis showed that Lys-73 is the major site of coupling. A mutant enzyme with a Lys-73----Gln replacement has a 50-fold reduced kcat/Km (with respect to tRNAAla) for aminoacylation but has a relatively small alteration of its kinetic parameters for ATP and alanine in the adenylate synthesis reaction. The data provide evidence that the 3' end of tRNAAla binds to a site in the enzyme domain responsible for adenylate synthesis and that a residue (Lys-73) in this domain is important for a tRNAAla-dependent step that is subsequent to the synthesis of the aminoacyladenylate intermediate.

Adenosine Triphosphate↗
PubMed1977

Aminoacylation of tRNA Trp from beef liver, yeast and E. coli by beef pancrease tryptophan-tRNA ligase. Stoichiometry of tRNATrp binding.

The Michaelis constants and the maximum velocities in the aminoacylation reaction of tRNATrp from beef liver, yeast and E. coli by pure beef pancreas tryptophan-tRNA ligase show that this mammalian enzyme recognizes and charges the two eucaryotic tRNAs with the same efficiency. The rate of aminoacylation of the procaryotic tRNATrp by the enzyme is three orders of magnitude lower. The pH optimum of aminoacylation is 8 for both eucaryotic tRNAs. The optimum magnesium concentration is different. The rate is maximum when magnesium concentration is stoichiometric to ATP concentration for tRNATrp from beef liver and 10 mM above ATP concentration for tRNATrp from yeast. The number of binding sites on the enzyme for the two eucaryotic tRNAs has been measured by equilibrium filtration on Sephadex G-100 and found equal to two.

Adenosine Triphosphate↗
PubMedDate not supplied

[Influence of the modification of Phe-tRNA synthetase from Escherichia coli by lysine- and arginine-specific reagent on the ionic interactions of the enzyme with tRNA Phe].

The influence of modification of Phe-RSase from E. coli MRE-600 by lysine- and arginine-specific reagent 2,4-pentandione on the Phe-RSase.tRNAPhe interactions was investigated. It was shown that modification of Phe-RSase with 2,4-pentandion leads to a decrease of the aminoacylation rate without any influence on the value of Km for tRNAPhe in this reaction and only a slight increase of the value of Kdiss for Phe-RSase.tRNAPhe complex. The log Km (Km-1)--ionic strength dependence for native enzyme and log Kdiss (K-1diss) for native enzyme and two forms modified on arginine and lysine residues were investigated. Results were interpreted quantitatively by Debye--Huckel approximation for two spherical macroions and by Daune approximation assuming that the region of tRNA implicated in ionic interactions is locally a cylindrical polyelectrolyte. It was shown that there are 2-4 electrostatic contacts in Phe-RSase.tRNAPhe interactions in limits of both approximations; modification of arginine residues in Phe-RSase doesn't change the number of electrostatic contacts, modification of lysine residues leads to an increase in the number of contacts. It was assumed that there are lysine residues in Phe-RSase essential for the tRNAPhe recognition. The possibility of participation of negative amino acid residues in electrostatic interactions with tRNAPhe is not excluded.

Amino Acyl-tRNA Synthetases↗
PubMedDate not supplied

[Interaction between tRNA-recognizing sites of phenylalanyl-tRNA synthetase from Escherichia coli MRE-600].

Modification of phenylalanyl-tRNA synthetase by N-Br-Ac-[14C]Phe-tRNAPhe results in two products, one being stable in alkaline conditions and the other being instable. The instable product is formed in the presence of an equimolar excess of the reagent over the enzyme while the stable one--in the presence of a tenfold excess. The experimental results are described by a kinetic model which takes into account the existence of two routes of modification of product formation. The first route is from the complex containing one molecule of the reagent and the other from the complex containing two molecules of the reagent. It is assumed that the presence of the molecule of the reagent in one center of the dimeric enzyme induces a change in the acceptor point of the other center, which leads to local conformational changes of the enzyme molecule interacting with the substrate analogue.

Amino Acyl-tRNA Synthetases↗
PubMed1997

A biologically active 53 kDa fragment of overproduced alanyl-tRNA synthetase from Thermus thermophilus HB8 specifically interacts with tRNA Ala acceptor helix.

The alaS gene encoding the alanyl-tRNA synthetase (AlaRS) from Thermus thermophilus HB8 was cloned and sequenced. The gene comprises 2646 bp, corresponding to 882 amino acids, 45% of which are identical to the enzyme from Escherichia coli . The T. thermophilus AlaRS was overproduced in E.coli , purified and characterized. It has high thermal stability up to approximately 65 degrees C, with a temperature optimum of aminoacylation activity at approximately 60 degrees C, and will be valuable for crystallization. The purified enzyme appears as a dimer with a specific activity of 220 U/mg and k cat/ K M values of 118 000/s/M for alanine and 114 000/s/M for ATP. By genetic engineering a 53 kDa fragment of AlaRS comprising the N-terminal 470 amino acids (AlaN470) was also overproduced and purified. It is as stable as entire AlaRS and sufficient for specific aminoacylation of intact tRNAAla, as well as acceptor stem microhelices with a G3-U70, but not U3-A70, I3-U70 or C3-U70, base pair. The reduced binding strength of such microhelices to AlaN470 enabled, due to the resulting fast exchange of the microhelices between free and complexed states, preliminary NMR analyses of the binding mode and intermolecular recognition.

Alanine-tRNA Ligase↗
PubMed2003

Variable sensitivity to bacterial methionyl-tRNA synthetase inhibitors reveals subpopulations of Streptococcus pneumoniae with two distinct methionyl-tRNA synthetase genes.

As reported previously (J. R. Jarvest et al., J. Med. Chem. 45:1952-1962, 2002), potent inhibitors (at nanomolar concentrations) of Staphylococcus aureus methionyl-tRNA synthetase (MetS; encoded by metS1) have been derived from a high-throughput screening assay hit. Optimized compounds showed excellent activities against staphylococcal and enterococcal pathogens. We report on the bimodal susceptibilities of S. pneumoniae strains, a significant fraction of which was found to be resistant (MIC, > or =8 mg/liter) to these inhibitors. Using molecular genetic techniques, we have found that the mechanism of resistance is the presence of a second, distantly related MetS enzyme, MetS2, encoded by metS2. We present evidence that the metS2 gene is necessary and sufficient for resistance to MetS inhibitors. PCR analysis for the presence of metS2 among a large sample (n = 315) of S. pneumoniae isolates revealed that it is widespread geographically and chronologically, occurring at a frequency of about 46%. All isolates tested also contained the metS1 gene. Searches of public sequence databases revealed that S. pneumoniae MetS2 was most similar to MetS in Bacillus anthracis, followed by MetS in various non-gram-positive bacterial, archaeal, and eukaryotic species, with streptococcal MetS being considerably less similar. We propose that the presence of metS2 in specific strains of S. pneumoniae is the result of horizontal gene transfer which has been driven by selection for resistance to some unknown class of naturally occurring antibiotics with similarities to recently reported synthetic MetS inhibitors.

Amino Acid Sequence↗
PubMed1982

A thermostable tRNA (guanosine-2')-methyltransferase from Thermus thermophilus HB27 and the effect of ribose methylation on the conformational stability of tRNA.

An S-adenosylmethionine-=dependent tRNA (guanosine-2'-)-methyltransferase (EC 2.1.1.34) was purified to the homogeneous state (2,400-fold) from a cell-free extract of an extreme thermophile, Thermus thermophilus HB27. The enzyme was highly resistant to heat as reported for other enzymes from thermophilic organism. The enzyme is monomeric and its molecular weight was estimated to be about 20,000. The Km values for S-adenosylmethionine and for Escherichia coli tRNAPhe were determined to be 0.47 microM and 10 nM, respectively, while the Ki for a competitive inhibitor S-adenosylhomocysteine, was 1.67 microM. When yeast tRNAPhe was methylated with the purified Gm-methyltransferase, a stoichiometric amount of methyl group was incorporated into the invariant guanosine at position 18 in the D-loop. Yeast tRNAPhe and E. coli tRNAMet, which were quantitatively methylated with the enzyme, were very similar to the native tRNAs with regard to amino acid acceptor activity and melting temperature, but were more resistant to RNase T1 and RNase A digestions than the corresponding native tRNAs.

Base Sequence↗
PubMed2003

Flexible adaptations in the structure of the tRNA-modifying enzyme tRNA-guanine transglycosylase and their implications for substrate selectivity, reaction mechanism and structure-based drug design.

The enzyme tRNA-guanine transglycosylase (TGT, EC 2.4.2.29) catalyses a base-exchange reaction that leads to anticodon modifications of certain tRNAs. The TGT enzymes of the eubacteria Zymomonas mobilis (Z. mobilis TGT) and Escherichia coli (E. coli TGT) show a different behaviour in the presence of competitive inhibitors. The active sites of both enzymes are identical apart from a single conservative amino acid exchange, namely Tyr106 of Z. mobilis TGT is replaced by a Phe in E. coli TGT. Although Tyr106 is, in contrast to Phe106, hydrogen bonded in the ligand-free structure, we can show by a mutational study of TGT(Y106F) that this is not the reason for the different responses upon competition. The TGT enzymes of various species differ in their substrate selectivity. Depending on the applied pH conditions and/or induced by ligand binding, a peptide-bond flip modulates the recognition properties of the substrate binding site, which changes between donor and acceptor functionality. Furthermore interstitial water molecules play an important role in these adaptations of the pocket. The flip of the peptide bond is further stabilised by a glutamate residue that operates as general acid/base. An active-site aspartate residue, presumed to operate as a nucleophile through covalent bonding during the base-exchange reaction, shows different conformations depending on the nature of the bound ligand. The induced-fit adaptations observed in the various TGT complex structures by multiple crystal-structure analyses are in agreement with the functional properties of the enzyme. In consequence, full understanding of this plasticity can be exploited for drug design.

Aspartic Acid↗
PubMed2005

Mechanism and substrate specificity of tRNA-guanine transglycosylases (TGTs): tRNA-modifying enzymes from the three different kingdoms of life share a common catalytic mechanism.

Transfer RNA-guanine transglycosylases (TGTs) are evolutionarily ancient enzymes, present in all kingdoms of life, catalyzing guanine exchange within their cognate tRNAs by modified 7-deazaguanine bases. Although distinct bases are incorporated into tRNA at different positions in a kingdom-specific manner, the catalytic subunits of TGTs are structurally well conserved. This review provides insight into the sequential steps along the reaction pathway, substrate specificity, and conformational adaptions of the binding pockets by comparison of TGT crystal structures in complex with RNA substrates of a eubacterial and an archaebacterial species. Substrate-binding modes indicate an evolutionarily conserved base-exchange mechanism with a conserved aspartate serving as a nucleophile through covalent binding to C1' of the guanosine ribose moiety in an intermediate state. A second conserved aspartate seems to control the spatial rearrangement of the ribose ring along the reaction pathway and supposedly operates as a general acid/base. Water molecules inside the binding pocket accommodating interaction sites subsequently occupied by polar atoms of substrates help to elucidate substrate-recognition and substrate-specificity features. This emphasizes the role of water molecules as general probes to map binding-site properties for structure-based drug design. Additionally, substrate-bound crystal structures allow the extraction of valuable information about the classification of the TGT superfamily into a subdivision of presumably homologous superfamilies adopting the triose-phosphate isomerase type barrel fold with a standard phosphate-binding motif.

Archaea↗
PubMed1982

Chemical probes for tRNA tertiary structure. Comparative alkylation of tRNA with methylnitrosourea, ethylnitrosourea and dimethylsulfate.

The tertiary structure of tRNA in solution can be proved by chemical modification experiments. Three reagents, N-ethyl-N-nitrosourea, N-methyl-N-nitrosourea and dimethylsulfate which are known to alkylate nucleic acids at nucleophilic centers were compared. It is found that N-ethyl-N-nitrosourea and N-methyl-N-nitrosourea mainly react with phosphate residues and dimethylsulfate only with the bases. With dimethylsulfate the extent of alkylation of guanosines is about one order of magnitude higher than that of the phosphates by the nitroso compounds.

Alkylating Agents↗
PubMed2004

The pre-tRNA nucleotide base and 2'-hydroxyl at N(-1) contribute to fidelity in tRNA processing by RNase P.

Fidelity in tRNA processing by the RNase P RNA from Escherichia coli depends, in part, on interactions with the nucleobase and 2' hydroxyl group of N(-1), the nucleotide immediately upstream of the site of RNA strand cleavage. Here, we report a series of biochemical and structure-function studies designed to address how these interactions contribute to cleavage site selection. We find that simultaneous disruption of cleavage site nucleobase and 2' hydroxyl interactions results in parallel reactions leading to correct cleavage and mis-cleavage one nucleotide upstream (5') of the correct site. Changes in Mg(2+) concentration and pH can influence the fraction of product that is incorrectly processed, with pH effects attributable to differences in the rate-limiting steps for the correct and mis-cleavage reaction pathways. Additionally, we provide evidence that interactions with the 2' hydroxyl group adjacent to the reactive phosphate group also contribute to catalysis at the mis-cleavage site. Finally, disruption of the adjacent 2'-hydroxyl contact has a greater effect on catalysis when pairing between the ribozyme and N(-1) is also disrupted, and the effects of simultaneously disrupting these contacts on binding are also non-additive. One implication of these results is that mis-cleavage will result from any combination of active site modifications that decrease the rate of correct cleavage beyond a certain threshold. Indeed, we find that inhibition of correct cleavage and corresponding mis-cleavage also results from disruption of any combination of active site contacts including metal ion interactions and conserved pairing interactions with the 3' RCCA sequence. Such redundancy in interactions needed for maintaining fidelity may reflect the necessity for multiple substrate recognition in vivo. These studies provide a framework for interpreting effects of substrate modifications on RNase P cleavage fidelity and provide evidence for interactions with the nucleobase and 2' hydroxyl group adjacent to the reactive phosphate group in the transition state.

Base Pairing↗
PubMed1999

Cysteinyl-tRNA formation: the last puzzle of aminoacyl-tRNA synthesis.

With the exception of the methanogenic archaea Methanococcus jannaschii and Methanobacterium thermoautotrophicum deltaH, all organisms surveyed contain orthologs of Escherichia coli cysteinyl-tRNA synthetase (CysRS). The characterization of CysRS-encoding (cysS) genes and the demonstration of their ability to complement an E. coli cysSts mutant reveal that Methanococcus maripaludis and Methanosarcina barkeri, two other methanogenic archaea, possess canonical CysRS proteins. A molecular phylogeny inferred from 40 CysRS sequences indicates that the CysRS of M. maripaludis and Methanosarcina spp. are specific relatives of the CysRS of Pyrococcus spp. and Chlamydia, respectively. This result suggests that the CysRS gene was acquired by lateral gene transfer in at least one euryarchaeotic lineage.

Amino Acid Sequence↗