Functional and structural studies on tRNA 2'-O-ribose methyltransferase, Bacillus subtilis CspR

Abstract

Transfer RNA undergoes extensive post-transcriptional modifications and one of the common modifications is the methylation of the 2’-OH group of ribose. In E.coli, TrmL acts as a 2’-O-methyltransferase by transferring a methyl group from SAM to the 2’-OH group of ribose at position 34 in EctRNALeuCAA and EctRNALeucmnm5UAA. In Bacillus subtilis, CspR is an ortholog of E.coli TrmL, sharing 45% sequence identity. However, until now, there has been no experimental validation of the properties of CspR regarding its function or structure. In this study, it is confirmed that CspR functions as a 2’-O-methyltransferase in B.subtilis at position 34 in BstRNALeuCAA through gene complementation and in vitro assay. Furthermore, it is verified that the activity of CspR depends on the presence of the N6-isopentenyl adenosine (i6A) modification at position 37 of the tRNA, which is catalyzed by MiaA. Additionally, we determined the crystal structure of B.subtilis CspR complexed with S-adenosyl-L-homocysteine (SAH), a reaction product. The structure revealed that BsCspR forms a dimeric conformation with a characteristic knot fold at the C-terminus, similar to other members of the SPOUT superfamily. Notably, the dimeric BsCspR exhibited a positively charged groove at the dimer interface, with both SAH molecules positioned nearby. This suggests a potential role of the dimer interface as a binding site for tRNA. Our study provides significant insights into the molecular basis underlying the properties of BsCspR.