tRNA Discrimination at the Binding Step by a Class II Aminoacyl-tRNA Synthetase

Abstract

Aminoacyl-tRNA synthetases preserve the fidelity of decoding genetic information by accurately joining amino acids to their cognate transfer RNAs. Here, tRNA discrimination at the level of binding by Escherichia coli histidyl-tRNA synthetase is addressed by filter binding, analytical ultracentrifugation, and iodine footprinting experiments. Competitive filter binding assays show that the presence of an adenylate analogue 5'-O-[N-(L-histidyl)sulfamoyl]adenosine, HSA, decreased the apparent dissociation constant (K(D)) for cognate tRNA(His) by more than 3-fold (from 3.87 to 1.17 microM), and doubled the apparent K(D) for noncognate tRNA(Phe) (from 7.3 to 14.5 microM). By contrast, no binding discrimination against mutant U73 tRNA(His) was observed, even in the presence of HSA. Additional filter binding studies showed tighter binding of both cognate and noncognate tRNAs by G405D mutant HisRS [Yan, W., Augustine, J., and Francklyn, C. (1996) Biochemistry 35, 6559], which possesses a single amino acid change in the C-terminal anticodon binding domain. Discrimination against noncognate tRNA was also observed in sedimentation velocity experiments, which showed that a stable complex was formed with the cognate tRNA(His) but not with noncognate tRNA(Phe). Footprinting experiments on wild-type versus G405D HisRS revealed characteristic alterations in the pattern of protection and enhancement of iodine cleavage at phosphates 5' to tRNA nucleotides in the anticodon and hinge regions. Together, these results suggest that the anticodon and core regions play major roles in the initial binding discrimination between cognate and noncognate tRNAs, whereas acceptor stem nucleotides, particularly at position 73, influence the reaction at steps after binding of tRNA.

The Gcd10p/Gcd14p complex is the essential two-subunit tRNA(1-methyladenosine) methyltransferase of Saccharomyces cerevisiae

“…Met by using a filter binding assay (12). The wild-type and FlagGcd14-3p͞Gcd10p mutant complexes bound tRNA i Met in a dose-dependent manner at levels far above the background level observed by using an equal amount of protein obtained from a mock purification using a strain containing untagged Gcd14p (Fig.…”

“…FlagGcd14p͞Gcd10p-tRNA complex formation was monitored by filter binding assay as described (12). Briefly, binding reactions (0.1 ml) were carried out in TB buffer (5 mM Tris⅐HCl, pH 7.5͞5 mM MgCl 2 ͞1.0 mM DTT͞25 mM NaCl), 50 g͞ml BSA, and 1 nM purified 32 P-labeled tRNA i Met from yJA146 at ambient temperature for 20 min with purified protein complexes at the concentrations indicated in Fig.…”

Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases

“…The chief shortcoming of the assay is that the optimal pH for capture of complexes on nitrocellulose is 5.5, which is well below the optimal pH for the aminoacylation reaction, and thus physiological conditions. Provided that all measurements are made at this pH, it is possible to make comparisons between different tRNA substrates and aaRS variants, because specificity for tRNA is retained at the lower pH [46,47]. Recently, this procedure was updated by Wong & Lohman [48], and later adapted for tRNA by Silvian & Steitz [49] into a 96-well format.…”

“…Recently, this procedure was updated by Wong & Lohman [48], and later adapted for tRNA by Silvian & Steitz [49] into a 96-well format. Conditions for the filter binding assay are provided in the previous references, as well as in a report describing the recognition of tRNA His by HisRS [47]. The updated method features a three layer membrane filter approach, each layer binding a different population of molecules.…”

PylSn and the Homologous N-terminal Domain of Pyrrolysyl-tRNA Synthetase Bind the tRNA That Is Essential for the Genetic Encoding of Pyrrolysine

“…where is the fractional saturation of tRNA-protein complex seen at the upper end of the PylSn concentrations tested (36). (Fig.…”