Structural insights into the mechanisms of SAM-dependent O-methyltransferases in bacterial tRNA wobble modifications

Abstract

Posttranscriptional modifications in tRNA anticodons are discovered across all life forms. Among these, the wobble position (the first nucleotide of anticodon) is one of the most heavily modified sites in tRNA, which bears an important role of codon recognition and preventing translational errors. Methyltransferases which function on the terminal oxygen of a modified wobble nucleotide are widely observed, studied, and attempted to determine their structure along with the molecules participating in their reactions. My work is composed in two parts: one covering E. coli CmoM (Part I), while the other is about B. subtilis CspR (Part II). These two enzymes are SAM dependent O-methyltransferases that operate on the tRNA wobble position. CmoM methylates 5-carboxymethoxyuridine at the terminal carboxylate oxygen to form a methyl ester (5-methoxycarbonylmethoxyuridine) at the wobble position. As a canonical methyltransferase which consists of the Rossmann-fold motif, the fundamentals of tRNA substrate recognition and molecular mechanism have been elusive. In this study, the CmoM-tRNASer complex structure reveals that enzyme-tRNA interactions are focused on the anticodon stem loop (ASL) region, and both macromolecules undergo local conformational changes. Accompanied site-directed mutagenesis assays support that CmoM seeks out 5-carboxymethoxyuridine at position 34 (cmo5U34) and G35 as crucial determinants to carry out its reaction. Alongside, tRNA features are highlighted including the flipping out of hypomodified uridine, cellularly-derived native modifications of tRNASer, and 16 nt-long variable arm. CspR, regarded as a 2’-O-methyltransferase modifying the wobble position ribose, had not been structurally revised nor biochemically investigated despite its orthology with E. coli TrmL. I provided evidence that B. subtilis CspR transfers a methyl group to the 2’-O-ribosyl position of tRNALeu containing C/U34 and i6A37 through gene complementation and in vitro biochemical analyses. In case of G34, m7G was formed in the presence of i6A37 when in vitro transcribed tRNA was used. For structural study, the SAH-bound CspR describes the cofactor binding site and topological features. In addition, the CspR-tRNA complex structure reveals that the tRNA anticodon loop region lies on the CspR’s dimeric interface, while the wobble nucleobase is not a determinant for recognition. Instead, residues A35 and i6A37 are recognized by the enzyme; especially the N6-isopentenyl group is encompassed by loops consisting of multiple hydrophobic residues that are disordered in the tRNA-free state. Collectively, my study provides insights into the structure-function relationship of wobble position modifying O-methyltransferases CmoM and CspR, and their specific roles in tRNA modification. CmoM catalyzes the formation of cmo5U34, with G35 acting as a determinant for enzyme catalysis. CspR's enzymatic function has been elucidated through biochemical and structural analyses, revealing A35 and i6A37 as critical determinants for its activity. These findings underscore the significance of wobble position methylations in biological processes and contribute to understanding tRNA modification enzymes.