Abstract

The outer membrane protein OmpA is a virulence factor in many mammalian pathogens. In previous global RNA structure probing studies, we found evidence for a temperature-modulated RNA structure in the 5'-untranslated region (5'-UTR) of the Yersinia pseudotuberculosis ompA transcript suggesting that opening of the structure at host-body temperature might relieve translational repression. Here, we support this hypothesis by quantitative reverse transcription PCR, translational reporter gene fusions, enzymatic RNA structure probing, and toeprinting assays. While ompA transcript levels decreased at 37°C compared to 25°C, translation of the transcript increased with increasing temperature. Biochemical experiments show that this is due to melting of the RNA structure, which permits ribosome binding to the 5'-UTR. A point mutation that locks the RNA structure in a closed conformation prevents translation by impairing ribosome access. Our findings add another common virulence factor to the growing list of pathogen-associated genes that are under RNA thermometer control.

Highlights

  • OmpA is a highly abundant and conserved outer membrane protein in Gram-negative bacteria (Smith et al, 2007)

  • OmpA forms complexes with the outer membrane protein Pal, which associates with the peptidoglycan layer via a conserved α-helical interaction motif (Clavel et al, 1998)

  • We identified a very promising RNA thermometer (RNAT) upstream of ompA by a global RNA structuromics approach, in which Y. pseudotuberculosis RNA structures were probed at three different temperatures (Righetti et al, 2016)

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Summary

Introduction

OmpA is a highly abundant and conserved outer membrane protein in Gram-negative bacteria (Smith et al, 2007). It has a barrel-like structure that confers porin activity. Apart from its function in the influx and efflux of various compounds, OmpA is a multifaceted protein with various other functions, its designation as a molecular Swiss army knife. It plays an important role in envelope stability. OmpA forms complexes with the outer membrane protein Pal, which associates with the peptidoglycan layer via a conserved α-helical interaction motif (Clavel et al, 1998)

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