A Proteomic and Transcriptomic Approach Reveals New Insight into β-methylthiolation of Escherichia coli Ribosomal Protein S12

Michael Brad Strader,Nina Costantino,Christopher A Elkins,Cai Yun Chen,Isha Patel,Anthony J Makusky,John S Choy,Donald L Court,Sanford P Markey,Jeffrey A Kowalak

doi:10.1074/mcp.m110.005199

Abstract

β-methylthiolation is a novel post-translational modification mapping to a universally conserved Asp 88 of the bacterial ribosomal protein S12. This S12 specific modification has been identified on orthologs from multiple bacterial species. The origin and functional significance was investigated with both a proteomic strategy to identify candidate S12 interactors and expression microarrays to search for phenotypes that result from targeted gene knockouts of select candidates. Utilizing an endogenous recombinant E. coli S12 protein with an affinity tag as bait, mass spectrometric analysis identified candidate S12 binding partners including RimO (previously shown to be required for this post-translational modification) and YcaO, a conserved protein of unknown function. Transcriptomic analysis of bacterial strains with deleted genes for RimO and YcaO identified an overlapping transcriptional phenotype suggesting that YcaO and RimO likely share a common function. As a follow up, quantitative mass spectrometry additionally indicated that both proteins dramatically impacted the modification status of S12. Collectively, these results indicate that the YcaO protein is involved in β-methylthiolation of S12 and its absence impairs the ability of RimO to modify S12. Additionally, the proteomic data from this study provides direct evidence that the E. coli specific β-methylthiolation likely occurs when S12 is assembled as part of a ribosomal subunit.

Highlights

Several studies have indicated that structural features of the ribosome including post-transcriptional and post-translational modifications occur on RNA and protein components, respectively
The highly conserved rimO gene displays striking similarity to the full-length gene sequence of the MiaB enzyme, a bifunctional system characterized in Escherichia coli that is involved in methylthiolation of transfer RNA (16 –19)
Creation and Utilization of SPA-S12 to Capture Molecular Complexes—In an effort to characterize the nature of ␤-methylthiolated S12 we adopted a sequence peptide affinity (SPA) strategy to identify candidate S12 interactors that are involved in ␤-methylthiolation or relevant to its biological function

Summary

Introduction

Several studies have indicated that structural features of the ribosome including post-transcriptional and post-translational modifications occur on RNA and protein components, respectively. The highly conserved rimO gene displays striking similarity to the full-length gene sequence of the MiaB enzyme, a bifunctional system characterized in Escherichia coli that is involved in methylthiolation of transfer RNA (tRNA) (16 –19). This tRNA modification involves addition of an S-methyl group to C2 of adenine 37 (A37) to produce 2-methylthio-N6-isopentenyl-adenosine (ms2i6A37) [1, 17]. Our goal was to gain insight into how RimO modifies S12 in vivo and to identify a potential function of this PTM Utilizing both proteomic and transcriptomic data we show that two candidate S12 interactors, RimO and YcaO, have a substantial effect on the methylthiolation status of the conserved Asp 88 in vivo and are likely to share a functional relationship. We present preliminary data that suggests a link between ␤-methylthiolation of S12 and certain effects on gene transcription

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Conclusion