Modular disordered domains autoregulate the RNA binding and annealing activities of the bacterial RNA chaperone Hfq

Hfq;sRNA;ChiX;intrinsically disordered;unstructured;RNA chaperone;autoregulation;small non-coding RNA;Rosetta;autoinhibition;post-transcriptional regulation;Biophysics

Santiago-Frangos, AndrewBarrick, Doug ;

Johns Hopkins University

关键词: Hfq;    sRNA;    ChiX;    intrinsically disordered;    unstructured;    RNA chaperone;    autoregulation;    small non-coding RNA;    Rosetta;    autoinhibition;    post-transcriptional regulation;    Biophysics;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/61051/SANTIAGO-FRANGOS-DISSERTATION-2018.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

The RNA chaperone Hfq is a bacterial Sm protein that stabilizes small regulatory RNAs (sRNAs) and helps them bind with their mRNA targets. Hfq and sRNAs regulate how bacteria respond to stress or changes in their environment and are involved in the virulence of a wide range of pathogenic and environmentally important bacteria. Like many RNA-binding proteins, Hfq consists of both structured, and unstructured components. While the RNA binding and annealing properties of the structured Sm core of Hfq has been well characterized, the function of the disordered C-terminal domain (CTD) has remained unclear. Here I use stopped flow spectroscopy to show that the CTD of Escherichia coli and Caulobacter crescentus Hfqs are not needed to accelerate RNA base pairing as had been previously proposed, but rather are required for the release of dsRNA. The Hfq CTD also mediates competition between sRNAs, offering a kinetic advantage to sRNAs that contact both the proximal and distal faces of the Hfq hexamer. This displacement allows Hfq to search among potential RNA partners and establishes a hierarchy of sRNA regulation. The change in sRNA hierarchy caused by deletion of the Hfq CTD in E. coli alters the sRNA accumulation and kinetics of sRNA regulation in vivo. Therefore, I propose that the Hfq CTD displaces sRNAs and annealed sRNA‧mRNA complexes from the Sm core, enabling Hfq to chaperone sRNA-mRNA interactions and rapidly cycle between competing targets in the cell.Rosetta modeling, competitive binding experiments and X-ray crystallography show that the acidic tips of E. coli and C. crescentus Hfq CTDs transiently bind the basic Sm core residues necessary for RNA annealing. The CTD tip competes against non-specific RNA binding, facilitates dsRNA release and prevents indiscriminate DNA aggregation, suggesting that this acidic peptide mimics nucleic acid to auto-regulate RNA binding to the Sm ring. The mechanism of CTD auto-inhibition I propose makes testable predictions of the function of Hfq in different bacterial genera and illuminates how Sm proteins may evolve new functions. Finally, the flexible CTDs appear to function as independent modules, enabling predictions for how Hfq homologs may act in different species of bacteria.