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

Abstract

Archaeal and eukaryotic tRNA (N 2 ,N 2 -guanine)-dimethyltransferase (Trm1) produces N 2 ,N 2 -dimethylguanine at position 26 in tRNA. In contrast, Trm1 from Aquifex aeolicus, a hyper-thermophilic eubacterium, modifies G27 as well as G26. Here, a gel mobility shift assay revealed that the T-arm in tRNA is the binding site of A. aeolicus Trm1. To address the multisite specificity, we performed an x-ray crystal structure study. The overall structure of A. aeolicus Trm1 is similar to that of archaeal Trm1, although there is a zinc-cysteine cluster in the C-terminal domain of A. aeolicus Trm1. The N-terminal domain is a typical catalytic domain of S-adenosyl-L-methionine-dependent methyltransferases. On the basis of the crystal structure and amino acid sequence alignment, we prepared 30 mutant Trm1 proteins. These mutant proteins clarified residues important for S-adenosyl-L-methionine binding and enabled us to propose a hypothetical reaction mechanism. Furthermore, the tRNA-binding site was also elucidated by methyl transfer assay and gel mobility shift assay. The electrostatic potential surface models of A. aeolicus and archaeal Trm1 proteins demonstrated that the distribution of positive charges differs between the two proteins. We constructed a tRNA-docking model, in which the T-arm structure was placed onto the large area of positive charge, which is the expected tRNA-binding site, of A. aeolicus Trm1. In this model, the target G26 base can be placed near the catalytic pocket; however, the nucleotide at position 27 gains closer access to the pocket. Thus, this docking model introduces a rational explanation of the multisite specificity of A. aeolicus Trm1.Methylation is one of the most common chemical modifications that occurs in a broad range of biomolecules, including nucleic acids, proteins, lipids, and small compounds. It is implicated in a variety of cellular processes, such as translation, transcription, processing of RNA, epigenetics, development, carcinogenesis, neurotransmission, cellular transport, infection, and bacterial host defense. Among the RNA species, methylated nucleotides appear in most noncoding RNAs, constituting more than half of the post-transcriptional modifications identified so far. In particular, tRNA contains abundant methylated nucleotides, which stabilize the L-shaped tRNA structure and improve their molecular recognition (1-3).Among the methylated nucleotides in tRNA, N 2 ,N 2

Substrate tRNA Recognition Mechanism of Eubacterial tRNA (m1A58) Methyltransferase (TrmI)

“…To confirm this, we performed a gel mobility shift analysis. As reported by Droogmans et al (20), it is much more difficult to perform gel mobility shift assays with TrmI than with other tRNA modification enzymes (53,54) because the large TrmI tetramer (more than 100 kDa) does not migrate into a normal polyacrylamide gel under electrophoresis. Consequently, we used agarose gels for the gel mobility shift assay as reported (20) and stained them with ethidium bromide.…”

Dynamics of RNA modification by a multi-site-specific tRNA methyltransferase

“…Nonetheless, there are a few RNA modifying enzymes that display a regional multi-site specificity, modifying consecutive positions in RNA. For instance, Aquifex aeolicus tRNA ( N 2, N 2-guanine)-dimethyltransferase Trm1 catalyzes the methylation not only of guanine 26 but also guanine 27 in tRNA ( 5 , 6 ). In Escherichia coli , the pseudouridine synthase TruA specifically modifies uridines at positions 38, 39 and/or 40 in the anticodon stem loop of tRNAs ( 7 ), while rRNA MTase KsgA catalyzes the dimethylation at the N6 position of two adjacent adenines A1518 and A1519 in the small subunit of ribosomal RNA ( 8 , 9 ).…”

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

“…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). Furthermore, the eukaryotic enzyme was identified from Saccharomyces cerevisiae as Trm2 (39 -41).…”