Structure-based Mechanism for m5C Formation by Trm4 from Saccharomyces cerevisiae

Abstract

The formation of 5-methylcytidine (m5C) is one of the well-conserved modifications across all RNA species. Trm4 is a tRNA-specific methyltransferase that was found in the baker’s yeast (Saccharomyces cerevisiae), which uses S-adenosyl-l-methionine as methyl donor to transfer a methyl group to the C5 of the cytidine at several positions of tRNA. During the reaction, it is known that the enzyme forms a covalently bonded intermediate by nucleophilic attack of Cys-310 on the C6 of cytosine. Cys-260 is considered to help release the product by acting as a general base, however its exact role is unknown. In an effort to obtain structure-guided insights into the precise enzymatic mechanism, we have constructed a series of truncated mutant proteins based on the prediction of the intrinsically unstructured region, and a site-directed mutant of Trm4 by replacing Cys-260 with alanine (C260A). In addition, we have tested the enzymatic activity of mutants with in vitro transcribed tRNA. Enzymatic formation of m5C was confirmed with wild-type (WT) Trm4 and Trm4 (28-684), whereas the product was not formed with Trm4 (28-418) and (46-684). C260A was purified as a complex of protein and RNA, which included m5C. These results revealed that the truncation of the specific region affects the function of the protein.