Abstract
Transfer RNA (Gm18) methyltransferase (TrmH) catalyzes methyl transfer from S-adenosyl-l-methionine to a conserved G18 in tRNA. We investigated the recognition mechanism of Thermus thermophilus TrmH for its guanosine target. Thirteen yeast tRNA(Phe) mutant transcripts were prepared in which the modification site and/or other nucleotides in the D-loop were substituted by dG, inosine, or other nucleotides. We then conducted methyl transfer kinetic studies, gel shift assays, and inhibition experiments using these tRNA variants. Sites of methylation were confirmed with RNA sequencing or primer extension. Although the G18G19 sequence is not essential for methylation by TrmH, disruption of G18G19 severely reduces the efficiency of methyl transfer. There is strict recognition of guanosine by TrmH, in that methylation occurs at the adjacent G19 when the G18 is replaced by dG or adenosine. The fact that TrmH methylates guanosine in D-loops from 4 to 12 nucleotides in length suggests that selection of the position of guanosine within the D-loop is relatively flexible. Our studies also demonstrate that the oxygen 6 atom of the guanine base is a positive determinant for TrmH recognition. The recognition process of TrmH for substrate is inducible and product-inhibited, in that tRNAs containing Gm18 are excluded by TrmH. In contrast, substitution of G18 with dG18 results in the formation of a more stable TrmH-tRNA complex. To address the mechanism, we performed the stopped-flow pre-steady state kinetic analysis. The result clearly showed that the binding of TrmH to tRNA is composed of at least three steps, the first bi-molecular binding and the subsequent two uni-molecular induced-fit processes.
Highlights
Modification of nucleic acids by methylation is a fundamental chemical process that can contribute to structure formation or alter gene expression patterns
A 5Ј-half-fragment synthesized with a deoxyguanosine 18 (dG18) substitution was ligated to a 3Ј-half-fragment, and successful ligation products were purified by gel electrophoresis (Fig. 2B, lane 5)
Based on our understanding of Transfer RNA (Gm18) methyltransferase (TrmH) catalysis, we predicted that tRNA-dG18 would not be methylated by TrmH
Summary
Materials—[methyl-14C]AdoMet (1.95 GBq/mmol) and [methyl-3H]AdoMet (2.47 TBq/mmol) were purchased from ICN. 3Ј-Half-fragments were transcribed by T7 RNA polymerase as reported previously [7] and purified using Qiagen plasmid mini kit column chromatography and 10% PAGE (7 M urea). To obtain kinetic parameters of TrmH for each tRNA, concentrations of TrmH and AdoMet were fixed at 0.1 and 37 M, respectively, whereas incubation times varied from 2 to 30 min depending on the methyl group acceptance activity for each transcript. Initial velocities of the reaction with 80 nM TrmH, 50 M [3H]AdoMet, and various concentrations (25, 50, 100, 200, 500, and 1000 nM) of wild-type yeast tRNAPhe transcript in 30 l of buffer B (50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, and 50 mM KCl) at 55 °C were compared in the absence or presence (25 or 50 nM) of methylated dG18 or dG18dG19A20 mutant tRNA transcript. To estimate the ratio of methylated tRNA to unmodified tRNA, methyl transfer filter assay was performed with [3H]AdoMet under the same conditions (7.7 M TrmH and tRNA)
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