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The C‐terminal region of thermophilic tRNA (m 7 G46) methyltransferase (TrmB) stabilizes the dimer structure and enhances fidelity of methylation
Author(s) -
Tomikawa Chie,
Ochi Anna,
Hori Hiroyuki
Publication year - 2007
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.21827
Subject(s) - aquifex aeolicus , transfer rna , biochemistry , alanine , amino acid , biology , methionine , chemistry , rna , escherichia coli , gene
Transfer RNA (m 7 G46) methyltransferase catalyzes methyl‐transfer from S‐adenosyl‐L‐methionine to N 7 atom of the semi‐conserved G46 base in tRNA. Aquifex aeolicus is a hyper thermophilic eubacterium that grows at close to 95°C. A. aeolicus tRNA (m 7 G46) methyltransferase [TrmB] has an elongated C‐terminal region as compared with mesophilic counterparts. In this study, the authors focused on the functions of this C‐terminal region. Analytic gel filtration chromatography and amino acid sequencing reveled that the start point (Glu202) of the C‐terminal region is often cleaved by proteases during purification steps and the C‐terminal region tightly binds to another subunit even in the presence of 6M urea. Because the C‐terminal region contains abundant basic amino acid residues, the authors assumed that some of these residues might be involved in tRNA binding. To address this idea, the authors prepared eight alanine substitution mutant proteins. However, measurements of initial velocities of these mutant proteins suggested that the basic amino acid residues in the C‐terminal region are not involved in tRNA binding. The authors investigated effects of the deletion of the C‐terminal region. Deletion mutant protein of the C‐terminal region (the core protein) was precipitated by incubation at 85°C, while the wild type protein was soluble at that temperature, demonstrating that the C‐terminal region contributes to the protein stability at high temperatures. The core protein had a methyl‐transfer activity to yeast tRNA Phe transcript. Furthermore, the core protein slowly methylated tRNA transcripts, which did not contain G46 base. Moreover, the modified base was identified as m 7 G by two‐dimensional thin layer chromatography. Thus, the deletion of the C‐terminal region causes nonspecific methylation of N 7 atom of guanine base(s) in tRNA transcripts. Proteins 2008. © 2007 Wiley‐Liss, Inc.