Functional Characterization of the YmcB and YqeV tRNA Methylthiotransferases of Bacillus Subtilis
Anton, Brian P.
Russell, Susan P.
Raleigh, Elisabeth A.
Limbach, Patrick A.
Roberts, Richard J.
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Citation (published version)Anton, Brian P., Susan P. Russell, Jason Vertrees, Simon Kasif, Elisabeth A. Raleigh, Patrick A. Limbach, Richard J. Roberts. "Functional characterization of the YmcB and YqeV tRNA methylthiotransferases of Bacillus subtilis" Nucleic Acids Research 38(18): 6195-6205. (2010)
Methylthiotransferases (MTTases) are a closely related family of proteins that perform both radical-S-adenosylmethionine (SAM) mediated sulfur insertion and SAM-dependent methylation to modify nucleic acid or protein targets with a methyl thioether group (–SCH3). Members of two of the four known subgroups of MTTases have been characterized, typified by MiaB, which modifies N6-isopentenyladenosine (i6A) to 2-methylthio-N6-isopentenyladenosine (ms2i6A) in tRNA, and RimO, which modifies a specific aspartate residue in ribosomal protein S12. In this work, we have characterized the two MTTases encoded by Bacillus subtilis 168 and find that, consistent with bioinformatic predictions, ymcB is required for ms2i6A formation (MiaB activity), and yqeV is required for modification of N6-threonylcarbamoyladenosine (t6A) to 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A) in tRNA. The enzyme responsible for the latter activity belongs to a third MTTase subgroup, no member of which has previously been characterized. We performed domain-swapping experiments between YmcB and YqeV to narrow down the protein domain(s) responsible for distinguishing i6A from t6A and found that the C-terminal TRAM domain, putatively involved with RNA binding, is likely not involved with this discrimination. Finally, we performed a computational analysis to identify candidate residues outside the TRAM domain that may be involved with substrate recognition. These residues represent interesting targets for further analysis.
RightsCopyright Anton, Brian P., Susan P. Russell, Jason Vertrees, Simon Kasif, Elisabeth A. Raleigh, Patrick A. Limbach, Richard J. Roberts2010. Published by Oxford University Press.