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: Based on these above investigations, residues from both the NTD (i.e. encompassing the SPOUT fold) and CTD are involved in tRNA binding to EcTrmJ. As expected, the isolated NTD and CTD alone could not bind to substrate tRNAs (Figure 7A). Moreover, a mixture of isolated NTD with half of the linker sequence (residues 1–170) and the CTD with the other half of the linker (residues 171–246) could not bind tRNAs (Figure 7A). Similarly, isolated NTD alone or together with CTD do not show any detectable methyltransferase activity (Figure 7B), although NTD alone could bind SAH as effectively as full-length EcTrmJ (Supplementary Figure S3).
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.