tRNA‐dependent amino acid discrimination by yeast seryl‐tRNA synthetase

Abstract

The ability of aminoacyl‐tRNA synthetases to distinguish between similar amino acids is crucial for accurate translation of the genetic code. Saccharomyces cerevisiae seryl‐tRNA synthetase (SerRS) employs tRNA‐dependent recognition of its cognate amino acid serine [Lenhard, B., Filipic, S., Landeka, I., Skrtic, I., Söll, D. & Weygand‐Durasevic, I. (1997) J. Biol. Chem.272, 1136–1141]. Here we show that dimeric SerRS enzyme complexed with one molecule of tRNASer is more specific and more efficient in catalyzing seryl‐adenylate formation than the apoenzyme alone. Sequence‐specific tRNA–protein interactions enhance discrimination of the amino acid substrate by yeast SerRS and diminish the misactivation of the structurally similar noncognate threonine. This may proceed via a tRNA‐induced conformational change in the enzyme's active site. The 3′‐terminal adenosine of tRNASer is not important in effecting the rearrangement of the serine binding site. Our results do not provide an indication for a readjustment of ATP binding in a tRNA‐assisted manner. The stoichiometric analyses of the complexes between the enzyme and tRNASer revealed that two cognate tRNA molecules can be bound to dimeric SerRS, however, with very different affinities.

The tRNA A76 Hydroxyl Groups Control Partitioning of the tRNA-dependent Pre- and Post-transfer Editing Pathways in Class I tRNA Synthetase

“…Pre-transfer editing can occur through enhanced dissociation of non-cognate aminoacyl-adenylate (2) or its enzymatic hydrolysis (1 and 3), which may be tRNA-independent (1) or tRNA-dependent (3). After transfer, misaminoacylated tRNA can be deacylated through post-transfer editing (4). and stored at Ϫ20°C.…”

“…We noticed that the exchanged tRNA is prone to instability, aggregation, and precipitation if dissolved in pure water and kept at Ϫ20°C. Therefore, it was stored in 5-10 mM HEPES at pH 7.5, and all kinetic assays contained 150 mM NH 4 4 Cl, 10 g/ml BSA, and 5 mM DTT. The reactions were stopped by adding 30 mM NaOAc, pH 4.5, and tRNA was extracted by acidic phenol, ethanol-precipitated, and dialyzed against 10 -20 mM NH 4 OAc, pH 5.0.…”

“…Therefore, it was stored in 5-10 mM HEPES at pH 7.5, and all kinetic assays contained 150 mM NH 4 4 Cl, 10 g/ml BSA, and 5 mM DTT. The reactions were stopped by adding 30 mM NaOAc, pH 4.5, and tRNA was extracted by acidic phenol, ethanol-precipitated, and dialyzed against 10 -20 mM NH 4 OAc, pH 5.0. Aminoacylated tRNAs were concentrated up to 50 mg/ml and further purified using reverse phase chromatography on Vydac C4 column (4.6 ϫ 250 mm; pore diameter, 300 Å; particle diameter, 5 m) to remove residual non-aminoacylated tRNA and nucleotides.…”

“…Aminoacylated tRNAs were concentrated up to 50 mg/ml and further purified using reverse phase chromatography on Vydac C4 column (4.6 ϫ 250 mm; pore diameter, 300 Å; particle diameter, 5 m) to remove residual non-aminoacylated tRNA and nucleotides. The column was equilibrated with buffer A (20 mM NH 4 OAc, pH 5.0, 10 mM Mg(OAc) 2 , 400 mM NaCl), and the retained molecules were eluted with the programmed linear gradients of buffer B (buffer A with 30% (v/v) ethanol). Fractions containing aminoacyl-tRNAs were pooled, ethanol-precipitated, and dialyzed against 1-2 mM NaOAc, pH 5.0.…”

Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases

“…The latter generally have lower affinity than protein:DNA interactions, so the complexes are less stable to the conditions of electrophoresis. In spite of these limitations, gel mobility shift analysis has been exploited to good effect, particularly in the seryl-tRNA synthetase system, where non-covalent aaRS:tRNA and covalently linked complexes have both been studied [50]. Unlike the filter binding assay, the gel mobility shift assay can be performed at pH values that are closer to the physiological pH of 7.5.…”