tRNA recognition by a bacterial tRNA Xm32 modification enzyme from the SPOUT methyltransferase superfamily.
Bottom Line: 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.
Affiliation: State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, The Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China University of Chinese Academy of Sciences, Beijing 100039, China.Show MeSH
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Mentions: In a previous study, it was shown that EcTrmJ requires full-length tRNA molecules as substrates, and that the identity of the D-stem and D-loop, but not the anticodon loop, is important for recognition by EcTrmJ (39). To obtain insights into the details of tRNA recognition by EcTrmJ, we furthered those studies by investigating the roles of the acceptor stem and elbow region. First, we truncated the acceptor stem in a step-wise manner (Figure 2A and Table 2) and, surprisingly, found that EcTrmJ could transfer the methyl group from SAM to those truncated tRNAs, unless the whole acceptor stem was completely deleted (Figure 2B). Then, we disrupted the L-shaped tertiary structure of tRNA by introducing single mutations into the elbow region (within D- and T-loops) of G18–U55 or G19–C56 (Figure 2A), which showed that all of these tRNA mutants could no longer be catalyzed by EcTrmJ (Figure 2C). Consistently, when we made double-mutations to restore the L-shape tertiary structure of tRNA by keeping the two hydrogen bonds in the U18–G55 region, the mutant tRNA could again be methylated by EcTrmJ (Figure 2C and Table 2). Overall, our results show that the L-shaped tertiary structure of tRNA is essential for recognition by EcTrmJ, while the acceptor stem region is not involved in this process.
Affiliation: State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, The Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China University of Chinese Academy of Sciences, Beijing 100039, China.