Characterization of Vibrio cholerae Hfq provides novel insights into the role of the Hfq C-terminal region.
Characterization of Vibrio cholerae Hfq provides novel insights into the role of the Hfq C-terminal region.
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DOI：
10.1016/j.jmb.2012.03.028
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发表时间：
2012-06-29
影响因子：
5.6
通讯作者：
Callaghan, Anastasia J.
中科院分区：
文献类型：
--
作者：
Vincent, Helen A.;Henderson, Charlotte A.;Ragan, Timothy J.;Garza-Garcia, Acely;Cary, Peter D.;Gowers, Darren M.;Malfois, Marc;Driscoll, Paul C.;Sobott, Frank;Callaghan, Anastasia J.
Hfq是细菌RNA结合蛋白，其促进小RNA介导的转录后基因调控。在霍乱弧菌中，Hfq和四个Hfq依赖的小RNA对于毒力基因的表达是必需的，但在分子水平上对这种机制知之甚少。为了更好地理解霍乱弧菌Hfq的结构和机制，我们使用生物化学和生物物理技术表征了该蛋白质，并与大肠杆菌Hfq进行比较。这两种蛋白的N端结构域（NTD）高度保守，但C端区域（CTR）在序列和长度上都不同。小角X-射线散射研究表明，这两种蛋白质采用星形六聚体结构，其中保守的NTD采用预期的Sm折叠，而可变的CTR是无序的，并从折叠的核心径向向外延伸。尽管它们的结构相似，但SDS-PAGE稳定性测定和碰撞诱导解离质谱显示，霍乱弧菌六聚体的稳定性低于大肠杆菌六聚体。杆菌我们认为这是由于NTD形成的亚基间界面与E. coli CTR来稳定该界面。然而，基于电泳迁移率变动分析，不同的CTR似乎确实执行关于RNA结合特异性的共同功能。总的来说，霍乱弧菌和大肠杆菌在基本性质上的异同是显而易见的。coli Hfq中进行组装和分子机制的研究。这是对霍乱弧菌Hfq的首次研究，Hfq是一种调节毒力基因的RNA结合蛋白。NTD与E. coli Hfq，CTR则不是。Cholesterol和E. coli Hfq CTR从六聚NTD核心向外延伸。Cholesterol和E. coli Hfq CTR增强Hfq的RNA结合特异性。只有E. coli CTR稳定Hfq六聚体。
Hfq is a bacterial RNA binding protein that facilitates small RNA-mediated posttranscriptional gene regulation. In Vibrio cholerae, 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 V. cholerae Hfq structure and mechanism, we characterized the protein, alongside Escherichia coli 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 V. cholerae hexamer is less stable than that of E. coli. We propose that this is due to minor differences between the intersubunit interface formed by the NTDs and the ability of the E. coli 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 V. cholerae and E. coli Hfq provide insight into their assembly and molecular mechanisms. ► This is the first study of V. cholerae Hfq, an RNA binding protein that regulates virulence genes. ► The NTD is homologous to E. coli Hfq, the CTR is not. ► Both V. cholerae and E. coli Hfq CTRs extend outwards from a hexameric NTD core. ► Both V. cholerae and E. coli Hfq CTRs enhance the RNA binding specificity of Hfq. ► Only the E. coli CTR stabilizes the Hfq hexamer.
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影响因子：
6.1
作者：
Konarev, PV;Volkov, VV;Svergun, DI
通讯作者：
Svergun, DI
影响因子：
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COMPTON, LA;JOHNSON, WC
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