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Substrate Specificity of the Radical SAM Methyl Synthase RlmN

  • Author(s): Fitzsimmons, Christina Marie
  • Advisor(s): Fujimori, Danica G
  • et al.
Abstract

Ribonucleic acids (RNA) are a large family of molecules that perform critical functions in the cell including coding the information of genes, regulation of gene expression, and facilitating and controlling translational accuracy. Post-transcriptional modifications help RNAs extend their function in cells by stabilizing correctly folded structures, avoiding misrecognition by other macromolecules, and preventing their own degradation. Although RNAs contain a wide variety of post-transcriptional marks, one of the most important is methylation, which accounts for nearly two-thirds of the distinct RNA modifications. Of particular interest is the bacterial radical SAM methyl synthase RlmN, which has the unusual ability to modify two distinct types of RNA: 23S rRNA and tRNA. In rRNA, RlmN installs a methyl group at the C2 position of A2503 of 23S rRNA, while in tRNA the modification occurs at nucleotide A37, immediately adjacent to the anticodon triplet. RNA modifying enzymes are generally highly specific for the type of RNA that they modify. Intrigued by the ability of RlmN to modify both rRNA and tRNA, we sought to determine the tRNA features necessary for methylation by RlmN. In this study, we utilized in vitro transcribed tRNAs, tRNA chimeras, and tRNA point mutants as model substrates to interrogate RNA recognition by RlmN, identifying position 38 of tRNAs as a critical determinant of substrate recognition. Following our biochemical experiments, we determined both the location and identity of an unknown minor species that was present in our reactions with the radical SAM methyl synthase RlmN. Herein we provide an outline of methods for labeling, digesting, and analyzing RNA oligos by LC-MS and LC-MS/MS. This approach allowed us to identify the location of the unknown modification at U54 in tRNA, and subsequent protein MS allowed us to determine the identity of the contaminating methyltransferase. Taken together, these biochemical and mass spectrometry techniques will contribute to the growing field of RNA modifications and RNA modifying proteins.

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