Characterization of Vibrio cholerae Hfq Provides Novel Insights into the Role of the Hfq C-Terminal Region

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• 作者：Helen A. Vincent¹ ; Charlotte A. Henderson¹ ; Timothy J. Ragan² ; Acely Garza-Garcia² ; Peter D. Cary¹ ; Darren M. Gowers¹ ; Marc Malfois³ ; Paul C. Driscoll² ; Frank Sobott⁴ ; ¹ ; Anastasia J. Callaghan¹ ; ^{anastasia.callaghan@port.ac.uk}
• 关键词：sRNA ; small RNA ; VcHfq ; Vibrio cholerae Hfq ; EcHfq ; Escherichia coli Hfq ; NTD ; N-terminal domain ; CTR ; C-terminal region ; SAXS ; small-angle X-ray scattering ; AUC ; analytical ultracentrifugation ; EMSA ; electrophoretic mobility shift assay ; Qrr ; quorum regu
• 刊名：Journal of Molecular Biology
• 出版年：2012
• 期刊代码：102_00222836
• 类别：imm
• 出版时间：29 June, 2012
• 卷：420
• 期：1-2
• 页码：56-69
• 文件大小：1397 K

摘要

Hfq is a bacterial RNA binding protein that facilitates small RNA-mediated posttranscriptional gene regulation. In m>Vibrio choleraem>, Hfq and four Hfq-dependent small RNAs are essential for the expression of virulence genes, but little is known about this mechanism at the molecular level. To better understand m>V. choleraem> Hfq structure and mechanism, we characterized the protein, alongside m>Escherichia colim> Hfq for comparison, using biochemical and biophysical techniques. The N-terminal domain (NTD) of the two proteins is highly conserved, but the C-terminal regions (CTRs) vary in both sequence and length. Small-angle X-ray scattering studies showed that both proteins adopt a star-shaped hexameric structure in which the conserved NTD adopts the expected Sm fold while the variable CTR is disordered and extends radially outwards from the folded core. Despite their structural similarity, SDS-PAGE stability assays and collision-induced dissociation mass spectrometry revealed that the m>V. choleraem> hexamer is less stable than that of m>E. colim>. We propose that this is due to minor differences between the intersubunit interface formed by the NTDs and the ability of the m>E. colim> CTR to stabilize this interface. However, based on electrophoretic mobility shift assays, the divergent CTRs do appear to perform a common function with regard to RNA-binding specificity. Overall, the similarities and differences in the fundamental properties of m>V. choleraem> and m>E. colim> Hfq provide insight into their assembly and molecular mechanisms.