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. 2008 May 13;105(19):6876-81.
doi: 10.1073/pnas.0802247105. Epub 2008 May 1.

Structure of a TrmA-RNA complex: A consensus RNA fold contributes to substrate selectivity and catalysis in m5U methyltransferases

Akram Alian  1 , Tom T Lee, Sarah L Griner, Robert M Stroud, Janet Finer-Moore
Affiliations

Structure of a TrmA-RNA complex: A consensus RNA fold contributes to substrate selectivity and catalysis in m5U methyltransferases

Akram Alian et al. Proc Natl Acad Sci U S A. .

Abstract

TrmA catalyzes S-adenosylmethionine (AdoMet)-dependent methylation of U54 in most tRNAs. We solved the structure of the Escherichia coli 5-methyluridine (m(5)U) 54 tRNA methyltransferase (MTase) TrmA in a covalent complex with a 19-nt T arm analog to 2.4-A resolution. Mutation of the TrmA catalytic base Glu-358 to Gln arrested catalysis and allowed isolation of the covalent TrmA-RNA complex for crystallization. The protein-RNA interface includes 6 nt of the T loop and two proximal base pairs of the stem. U54 is flipped out of the loop into the active site. A58 occupies the space of the everted U54 and is part of a collinear base stack G53-A58-G57-C56-U55. The RNA fold is different from T loop conformations in unbound tRNA or T arm analogs, but nearly identical to the fold of the RNA loop bound at the active site of the m(5)U MTase RumA. In both enzymes, this consensus fold presents the target U and the following two bases to a conserved binding groove on the protein. Outside of this fold, the RumA and TrmA substrates have completely different structures and protein interfaces. Loop residues other than the target U54 make more than half of their hydrogen bonds to the protein via sugar-phosphate moieties, accounting, in part, for the broad consensus sequence for TrmA substrates.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Mechanism and substrates of TrmA. (A) The proposed catalytic mechanism of RNA m5U Mtases (4). (B) Consensus sequence of the T arm of tRNA derived from the sequences of E. coli tRNAs known to be methylated (10). Blue indicates conserved and yellow indicates nonconserved bases in the T arm. The target uridine is red. The sequence of the T arm used in this study is indicated next to the base numbering. Circles indicate the additional base pair added to the original T loop sequence. 2′-Se-Me-U is indicated as Use. (C) Side view of TrmA–T arm structure.
Fig. 2.
Fig. 2.
Sequence alignment of TrmA and RumA. Conserved residues are white on a red background, and conservative substitutions are red on a white background. The secondary structures of TrmA and RumA are shown above and below their sequences, respectively (coil = helix, arrow = β-strand, T = turn) and are colored purple, yellow, and blue for the cross-over segment, RNA binding, and catalytic domains, respectively. Conserved motifs are marked with blue dashes under the sequence alignment.
Fig. 3.
Fig. 3.
Cartoon of superimposed TrmA and RumA structures. RumA is the background structure (green). The TrmA color scheme is as in Fig. 2. The m5U MTase motifs are red. The Cα of the catalytic sulfhydryl is shown as a red sphere. (Inset) A close-up of the AdoMet binding site, with AdoHcy from RumA complex in violet.
Fig. 4.
Fig. 4.
Substrate binding in TrmA and RumA. (A) Comparison of loop conformations of the T-arm analog from the TrmA–RNA structure (pink and red), with the T loop of unbound tRNAPhe (orange) (13). U54 is shown in stick representation. (B) Comparison of the 5′-loop conformation of the 37-mer in the RumA–RNA cocrystal structure (blue) with the conformation this fragment has in the mature ribosome (orange) (8). U1939 is shown as sticks. (C) Overlap of the RNA loops bound to the active sites of TrmA (pink, bases 53–58) and RumA (blue, bases 1938–1942). (D) (Left) Surface representation of TrmA bound to its 19-mer substrate (magenta), with the RumA 37-mer substrate superposed in the yellow cartoon. (Right) Surface representation of RumA bound to its substrate (yellow), with the TrmA substrate superposed (magenta). RNA binding, catalytic, and OB-fold domains are colored blue, gray, and green, respectively.
Fig. 5.
Fig. 5.
RNA–protein interactions. (A) Diagram of the secondary structure and interactions of the bound RNA. (B) Stereoview of a stick representation of T-loop bases U54, U55, C56, and G57 bound to the TrmA active site. Putative hydrogen bonds between the target U and the protein are shown with red dotted lines, and hydrogen bonds between the other bases and the protein are shown with green dotted lines. (C) Close-up of the interactions between the target uridine and the amino acid side chains overlaid with Fo − Fc map (4.5 σ) calculated with U54 and surrounding side chains omitted.
Fig. 6.
Fig. 6.
Crossed-eyes stereoview of D loop–T loop interface of a TrmA–tRNA model made by aligning the tRNA T stem with the 19-mer stem in the crystal structure. Protein residues are blue, and tRNA bases are red, except for U17, G18, and G19, which are yellow.
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