tRNA recognition by a bacterial tRNA Xm32 modification enzyme from the SPOUT methyltransferase superfamily

Abstract

TrmJ proteins from the SPOUT methyltransferase superfamily are tRNA Xm32 modification enzymes that occur in bacteria and archaea. Unlike archaeal TrmJ, bacterial TrmJ require full-length tRNA molecules as substrates. It remains unknown how bacterial TrmJs recognize substrate tRNAs and specifically catalyze a 2'-O modification at ribose 32. Herein, we demonstrate that all six Escherichia coli (Ec) tRNAs with 2'-O-methylated nucleosides at position 32 are substrates of EcTrmJ, and we show that the elbow region of tRNA, but not the amino acid acceptor stem, is needed for the methylation reaction. Our crystallographic study reveals that full-length EcTrmJ forms an unusual dimer in the asymmetric unit, with both the catalytic SPOUT domain and C-terminal extension forming separate dimeric associations. Based on these findings, we used electrophoretic mobility shift assay, isothermal titration calorimetry and enzymatic methods to identify amino acids within EcTrmJ that are involved in tRNA binding. We found that tRNA recognition by EcTrmJ involves the cooperative influences of conserved residues from both the SPOUT and extensional domains, and that this process is regulated by the flexible hinge region that connects these two domains.

Figure 1.

The tRNA substrates of TrmJ in Escherichia coli. (A) The cloverleaf structures of six E. coli tRNAs with a 2′-O-methylated ribose in position 32 from the MODOMICS database. (B and C) The methyltransferase activity of EcTrmJ for tRNA substrates. Error bars represent standard errors of three independent experiments.

Figure 2.

Recognition of tRNA elements by EcTrmJ. (A) A model of L-shaped EctRNA_f^Met₁ with arrows showing truncations on the acceptor stem. (B) The methyltransferase activity of EcTrmJ for EctRNA_f^Met₁ and its various truncations. (C) The methyltransferase activity of EcTrmJ for EctRNA_f^Met₁ and its mutations on the elbow region. Error bars represent standard errors of three independent experiments.

Figure 3.

The overall structure of the EcTrmJ–SAH complex and a sequence alignment of bacterial TrmJs. (A) A ribbon diagram shows the overall structure of EcTrmJ in complex with SAH (presented in spheres). The structure is shown as a dimer, with one subunit in green and the other one in cyan. The invisible linker regions are shown as dotted lines. (B) An overlay of the two monomers (cyan and green) in the asymmetric EcTrmJ dimer that superposition the SPOUT domains alone. The spheres represent bound SAH. (C) A structure-based multiple amino acid sequence alignment of bacterial TrmJs from model organisms. Abbreviations used: Ec, Escherichia coli; Hi, Haemophilus influenza; Yp, Yersinia pestis; Cj, Cellvibrio japonicas; Cs, Cyanobacteriumstanieri; and Bs, Bacillus subtilis. The secondary structure elements of EcTrmJ are labeled above the alignment. Residues involved in tRNA binding that were identified in this study are marked by a black pentagon.

Figure 4.

Structural details of the EcTrmJ–SAH complex. SAH bound in one subunit of EcTrmJ (A) and all residues within 4 Å from SAH are shown as a stick (B). The carbon atom of SAH is shown in orange, the backbone of EcTrmJ and the SAH-binding pocket are shown in green and salmon, respectively, and part of the loop β4–α4 that contained residues R82 and R84 is shown in magenta. (C) A ribbon diagram showing the CTD dimer, residues at the dimer interface are shown as sticks. (D) The methyltransferase activities of various EcTrmJ mutants. Error bars represent standard errors of three independent experiments. (E) The vacuum electrostatics of EcTrmJ; two large positively charged patches are circled in green.

Figure 5.

Ala mutations of basic amino acid residues near the SAM/SAH-binding pocket of EcTrmJ. (A) The binding affinities of EcTrmJs for EctRNA_f^Met₁ were analyzed using a gel mobility shift assay. (B) Methyltransferase activities of various EcTrmJ mutants. Error bars represent standard errors of three independent experiments. (C) The SAH binding affinity, as measured by ITC.

Figure 6.

Ala mutations of protein surface basic amino acid residues in the CTD of EcTrmJ. (A) The binding affinities of EcTrmJs for EctRNA_f^Met₁ were analyzed by EMSA. (B) The methyltransferase activities of EcTrmJ variants. Error bars represent standard errors of three independent experiments. (C) The binding affinities for SAH by ITC.

Figure 7.

Roles of the hinge region that connect the NTD and CTD. (A) The tRNA binding affinities of the isolated NTD and CTD were analyzed by EMSA. (B) The methyltransferase activities of the isolated NTD and CTD. (C and D) The methyltransferase activities of EcTrmJ variants in the hinge region. The NTD and CTD in this figure are domains with residues from 1–170 and 171–246, respectively. Error bars represent standard errors of three independent experiments.

Figure 8.

A proposed model of tRNA binding to EcTrmJ. (A and B) Proposed models of the EcTrmJ dimer bound with one tRNA substrate in the same view; the tRNA backbone is shown in orange.