Conformation and mechanical property of rpoS mRNA inhibitory stem studied by optical tweezers and X-ray scattering.

Xinyao Hu,Lingna Yang,Yunyu Shi,Xuanling Li,Yilin Zhu,Yinmei Li,Qingguo Gong,Haowei Wang,Yong-Bin Yan

doi:10.1371/journal.pone.0222938

Xinyao Hu, Lingna Yang + Show 7 more

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https://doi.org/10.1371/journal.pone.0222938

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Abstract

3′ downstream inhibitory stem plays a crucial role in locking rpoS mRNA 5' untranslated region in a self-inhibitory state. Here, we used optical tweezers to study the unfolding/refolding of rpoS inhibitory stem in the absence and presence of Mg2+. We found adding Mg2+ decreased the free energy of the RNA junction without re-arranging its secondary structure, through confirming that this RNA formed a canonical RNA three-way junction. We suspected increased free energy might change the relative orientation of different stems of rpoS and confirmed this by small angle X-ray scattering. Such changed conformation may improve Hfq-bridged annealing between sRNA and rpoS RNA inhibitory stem. We established a convenient route to analyze the changes of RNA conformation and folding dynamics by combining optical tweezers with X-ray scattering methods. This route can be easily applied in the studies of other RNA structure and ligand-RNA.

Highlights

Bacteria developed a global stressors response to various environmental stress including high temperature, starvation, osmotic pressure, and unsuitable pH during life cycle [1]
RpoS mRNA is normally in a suppressed state, in which the rpoS mRNA 5’ untranslated region (5’-UTR) Shine-Dalgarono (SD) sequence is folded inside an inhibitory stem[5, 6]
We showed that the combination of optical tweezers and Small angle X-ray scattering (SAXS) is a useful strategy to explore the structure-and-dynamics of RNA molecules, especially when their high-resolution three-dimensional structure are not available

Summary

Introduction

Bacteria developed a global stressors response to various environmental stress including high temperature, starvation, osmotic pressure, and unsuitable pH during life cycle [1]. We measured the free energy change corresponding to the conformational change of rpoS RNA with optical tweezers, and dissected the RNA structure from the single molecule level. We analyzed the structure and folding/unfolding kinetics of rpoS mRNA inhibitory stem, by combination of optical tweezers results with overall RNA topology obtained by SARX.

Results

Conclusion