Interactions between aminoacyl tRNA synthetases, tRNAs, and fluorescent dyes (original) (raw)
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European journal of biochemistry / FEBS, 1976
By following the tryptophan fluorescence of yeast seryl-tRNA synthetase on addition of tRNA Ser it was observed that the number of binding sites for tRNA decreases from two to one with increasing temperature, ATP or KCl concentration. Concomitantly a considerable decrease of the apparent binding constant was observed. The variation in the number of binding sites is explained by the presence of at least one temperature and ionic strength sensitive binding site and one temperature and ionic strength independent binding site. Relaxation kinetic experiments revealed two binding processes: a fast one depending on tRNA concentration and ionic strength and a slow one, which appeared to be independent of tRNA concentration and ionic strength. Enzyme kinetic studies showed that the activity of seryl-tRNA synthetase strongly depends on the KCl concentration and exhibits a maximum at 0.2 M KCl. Based on the data from relaxation and enzyme kinetic experiments a model is suggested for the recogn...
Nucleic Acids Research, 1995
We describe a convenient, simple and novel continuous spectrophotometric method for the determination of aminoacyl-tRNA synthetase activity. The assay relies upon the measurement of inorganic pyrophosphate generated in the first step of the aminoacylation of a tRNA. Pyrophosphate release is coupled to inorganic pyrophosphatase, to generate phosphate, which in turn is used as the substrate of purine nucleoside phosphorylase to catalyze the N-glycosidic cleavage of 2-amino 6-mercapto 7-methylpurine ribonucleoside. Of the reaction products, ribose 1-phosphate and 2-amino 6-mercapto 7-methylpurine, the latter has a high absorbance at 360 nm relative to the nucleoside and hence provides a spectrophotometric signal that can be continuously followed. The nondestructive nature of the spectrophotometric assay allowed the re-use of the tRNAs in question in successive experiments. The usefulness of this method was demonstrated for glutaminyl-tRNA synthetase (GInRS) and tryptophanyl-tRNA synthetase. Initial velocities measured using this assay correlate closely with those assayed by quantitation of [3H]Gln-tRNA or [14C]Trp-tRNA formation respectively. In both cases amino acid transfer from the aminoacyl adenylate to the tRNA represents the rate determining step. In addition, aminoacyl adenylate formation by aspartyl-tRNA synthetase was followed and provided a more sensitive means of active site titration than existing techniques. Finally, this novel method was used to provide direct evidence for the cooperativity of tRNA and ATP binding to GInRS.
Equivalent and Nonequivalent Binding Sites for tRNA on Aminoacyl-tRNA Synthetases
European Journal of Biochemistry, 1975
Complexes between tRNAPhe (yeast), tRNAser (yeast) and tRNATy' (Escherichia coli) and their cognate aminoacyl-tRNA synthetases have been studied by sedimentation velocity runs in an analytical ultracentrifuge. The amount of complex formation was determined by the absorption and the sedimentation coefficients of the fast-moving boundary in the presence of excess tRNA or excess synthetase respectively. The same method has been applied to unspecific combinations of tRNAs and synthetases. Inactive material of tRNA or synthetase does not influence the results.
A Spectrophotometric Assay for Quantitative Measurement of Aminoacyl-tRNA Synthetase Activity
Journal of Biomolecular Screening, 2013
Aminoacyl-tRNA synthetases are enzymes that charge specific tRNAs with their cognate amino acids and play an essential role in the initial steps of protein synthesis. Because these enzymes are attractive targets for drug development in many microorganisms, there is a pressing need for assays suitable for compound screening. We developed (1) a high throughput assay for measuring aminoacyl-tRNA synthetase activity and (2) an accompanying method for preparing the tRNA substrate. The assay can be performed in 96-well plates and relies on malachite green detection of pyrophosphate (Pi) as an indicator of aminoacyl-tRNA synthetase activity. Analysis of Trypanosoma brucei isoleucyl-tRNA synthetase (IleRS) activity showed that the assay exhibits sensitivity to picomoles of product, and yielded a Z′ factor of 0.56. We show that this assay is applicable to other aminoacyl-tRNA synthetases and to enzyme inhibition studies. Using this assay, we found that the compound NSC616354 inhibits recombinant IleRS with an IC 50 of 0.6 µM. Enzymology studies were also performed with rIleRS and its K m and k cat determined as 3.97 x 10 -5 mol/L and 142 S -1 , respectively. This assay will facilitate the screening of compounds to identify inhibitors of aminoacyl-tRNA synthetases.
Proceedings of the National Academy of Sciences, 1988
Analysis of the in vivo amber suppressor activity of mutants derived from two Escherichia coli serine tRNAs shows that substitution of 2 base pairs in the acceptor helix changes a serine suppressor tRNA to an efficient glutamine acceptor. Determination of the amino acid inserted in vivo into protein by this tRNA shows that these changes reduce the tRNA recognition by seryl-tRNA synthetase while increasing that of glutaminyl-tRNA synthetase. This implies that misaminoacylation in vivo is dependent on the competition by different synthetases for the tRNA. In addition, the "translational efficiency" of tRNA is an integral part in observing misaminoacylation in vivo.
Biochemistry, 1982
Arginyl- and lysyl-tRNA synthetases copurify throughout a six-step chromatographic procedure resulting in a purification of 605- and 559-fold, respectively. The purified enzymes were estimated to be 98% pure with a stoichiometry of 1:1 from acrylamide gel electrophoresis under denaturing conditions. On the basis of a native molecular weight of 285000 calculated from s20,w, Rs, and V and subunit molecular weights of 73000 and 65000 obtained by sodium dodecyl sulfate gel electrophoresis, the synthetases appear to exist as a tetramer. The tetrameric structure was also supported by cross-linking studies. These results are consistent with an alpha 2 beta 2 structure, but an alpha beta structure has not been ruled out.