Probing the conformational changes of in vivo overexpressed cell cycle regulator 6S ncRNA

Eleni Makraki; Sophia Miliara; Michalis Pagkalos; Michael Kokkinidis; Efstratios Mylonas; Vasiliki E Fadouloglou

doi:10.3389/fmolb.2023.1219668

Probing the conformational changes of in vivo overexpressed cell cycle regulator 6S ncRNA

Front Mol Biosci. 2023 Jul 17:10:1219668. doi: 10.3389/fmolb.2023.1219668. eCollection 2023.

Authors

Eleni Makraki¹, Sophia Miliara¹, Michalis Pagkalos^{1

2}, Michael Kokkinidis^{1

2}, Efstratios Mylonas¹, Vasiliki E Fadouloglou^{1

3}

Affiliations

¹ Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas (IMBB-FORTH), Heraklion, Greece.
² Department of Biology, University of Crete, Heraklion, Greece.
³ Department of Molecular Biology and Genetics, Democritus University of Thrace, Komotini, Greece.

Abstract

The non-coding 6S RNA is a master regulator of the cell cycle in bacteria which binds to the RNA polymerase-σ⁷⁰ holoenzyme during the stationary phase to inhibit transcription from the primary σ factor. Inhibition is reversed upon outgrowth from the stationary phase by synthesis of small product RNA transcripts (pRNAs). 6S and its complex with a pRNA were structurally characterized using Small Angle X-ray Scattering. The 3D models of 6S and 6S:pRNA complex presented here, demonstrate that the fairly linear and extended structure of 6S undergoes a major conformational change upon binding to pRNA. In particular, 6S:pRNA complex formation is associated with a compaction of the overall 6S size and an expansion of its central domain. Our structural models are consistent with the hypothesis that the resultant particle has a shape and size incompatible with binding to RNA polymerase-σ⁷⁰. Overall, by use of an optimized in vivo methodological approach, especially useful for structural studies, our study considerably improves our understanding of the structural basis of 6S regulation by offering a mechanistic glimpse of the 6S transcriptional control.

Keywords: 6S RNA; SAXS; in vivo RNA overexpression; non-coding RNA; pRNAs.

Grants and funding

This work was supported by the Marie Curie Reintegration Grant FP7-PEOPLE-2010-RG (PERG08-GA-1020-276861), the action “Supporting Postdoctoral Researchers” (NSRF 2007-2013 LS1/3258), the EU-FP7 REGPOT InnovCrete program, a grant from the Hellenic Foundation for Research and Innovation (HFRI) (2nd call for Postdocs proposal 00088) and the project “InTechThrace: Integrated Technologies in biomedical research: multilevel biomarker analysis in Thrace” (MIS Code 5047285), under the Operational Program “Competitiveness, Entrepreneurship & Innovation” (EPAnEK), co-funded by the European Regional Development Fund (ERDF) and national resources (Partnership Agreement 2014-2020).