Posttranscriptional modifications of RNA bases have a profound impact on the biological function of RNA. RlmN is a recently discovered and mechanistically novel radical SAM methyl synthase with a unique ability to modify both ribosomal RNA (rRNA) and transfer RNA (tRNA). This enzyme methylates the C2 amidine carbon of an adenosine moiety. Adenosine 2503, the site of modification in 23S rRNA, is located at the entrance of the nascent peptide exit tunnel of the bacterial ribosome. A subset of tRNAs also contain a C2 methyl adenosine, A37, located immediately 3' to the anticodon. The location of these modifications in two functionally important regions of RNA indicates their significance in bacterial transcriptional regulation. In order to better understand the ability of this enzyme to modify different types of RNA, we sought to determine the features of RNA that are required for methylation by RlmN. We generated a number of point mutant and chimeric tRNA constructs to determine the nucleotide sequence and secondary structural features required for methylation. Our results indicate that the anticodon stem-loop and the variable loop of tRNA are important for recognition. Additionally, deuterium kinetic isotope effect experiments provide evidence for the role of the RNA substrate in organizing the active site during catalysis. Our experiments point to the importance of substrate recognition in initiating methylation by RlmN.

Substrate specificity of the radical SAM methyl synthase RlmN

248th National Meeting (2014)

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