Flexible Recognition of the tRNA G18 Methylation Target Site by TrmH Methyltransferase through First Binding and Induced Fit Processes

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 presteady 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.

Unexpected expansion of tRNA substrate recognition by the yeast m1G9methyltransferase Trm10

“…S1; Table 1). Precise determination of the K M,tRNA for each tRNA substrate was difficult because of significant product inhibition observed in the time courses, likely due to inhibition by the S-adenosylhomocysteine (SAH) product of the methyltransferase reaction, as is frequently observed with other tRNA methyltransferases (Shugart 1978;Hjalmarsson et al 1983;Ochi et al 2010). Nonetheless, by restricting measurements of initial rate to <5% of product formation, where the product inhibition was not as evident, we were able to measure observed rates that agreed well (within threefold), even when measured on different days.…”

The tRNA Recognition Mechanism of Folate/FAD-dependent tRNA Methyltransferase (TrmFO)

“…Gel Mobility Shift Assay-The gel mobility shift assay was performed as described in a previous report (31 Phe transcript (25 g) was individually methylated by TrmH (5 g), TrmB (7.5 g), and TrmI (7 g) in the presence of 200 M AdoMet for 3 h at 55°C. In the case of ⌿55 modification, the transcript (25 g) was incubated with TruB (7.5 g) without AdoMet for 3 h at 55°C.…”

“…To address this issue, we performed a gel mobility shift assay, which has been Phe was weak, suggesting that this tRNA is excluded before the structural change process or in the early stage of the structural change process. previously used for binding assays of several modification enzymes (10,31,39). As shown in Fig.…”

“…For example, the local RNA structure recognition is commonly observed in tRNA (⌿55) synthase (28), tRNA guanine transglycosidase (29), tRNA (Gm18) methyltransferase (30,31), tRNA (m 1 G37) methyltransferase (32), tRNA (m 7 G46) methyltransferase (33), tRNA (i 6 A37) isopentenyltransferase (34,35), and others. The single displacement methyl transfer mechanism is common to at least tRNA (m (36,37) and tRNA (Gm18) methyltransferase (38,39).…”

Substrate tRNA Recognition Mechanism of a Multisite-specific tRNA Methyltransferase, Aquifex aeolicus Trm1, Based on the X-ray Crystal Structure

“…RNA transcripts were prepared as reported previously (26). If discrimination between the m 2 G and m 2 2 G formation activities was necessary, we employed two-dimensional thin layer chromatography as described in our previous reports (21,27 Gel Mobility Shift Assay-A gel mobility shift assay was performed according to our previous reports (30,31).…”