"The catalytic domain of topological knot tRNA methyltransferase (TrmH) discriminates between substrate tRNA and non-substrate tRNA via an induced-fit process."

Ochi A, Makabe K, Yamagami R, Hirata A, Sakaguchi R, Hou YM, Watanabe K, Nureki O, Kuwajima K, Hori H...



Published 2013-07-18 in J Biol Chem .

Pubmed ID: 23867454
DOI identifier: -

Abstract:
A conserved guanosine at position 18 (G18) in the D-loop of tRNAs is often modified to 2'-O-methylguanosine (Gm). Formation of Gm18 in eubacterial tRNA is catalyzed by tRNA (Gm18) methyltransferase (TrmH). TrmH enzymes can be divided into two types based on their substrate tRNA specificity. Type I TrmH including Thermus thermophilus TrmH can modify all tRNA species, whereas Type II TrmH, for example Escherichia coli TrmH, modifies only a subset of tRNA species. Our previous crystal study showed that T. thermophilus TrmH is a Class IV AdoMet-dependent methyltransferase, which maintains a topological knot structure in the catalytic domain. Because TrmH enzymes have short stretches at the N- and C-termini instead of a clear RNA binding domain, these stretches are believed to be involved in tRNA recognition. In the current study, we demonstrate by site-directed mutagenesis that both N- and C-terminal regions function in tRNA binding. However, in vitro and in vivo chimera protein studies, in which four chimeric proteins of Type I and II TrmHs were used, demonstrated that the catalytic domain discriminates substrate tRNAs from non-substrate tRNAs. Thus, the N- and C-terminal regions do not function in the substrate tRNA discrimination process. Pre-steady state analysis of complex formation between mutant TrmH proteins and tRNA by stopped-flow fluorescence measurement revealed that the C-terminal region works in the initial binding process, in which non-substrate tRNA is not excluded, and that structural movement of the motif 2 region of the catalytic domain in an induced-fit process is involved in substrate tRNA discrimination.


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Last modification of this entry: Aug. 5, 2013