Defining the structural mechanisms of Hfq binding to cognate nucleic acids

描述(申请人提供)：Hfq是一种在许多细菌中发现的多效性转录后调节因子。Hfq在细胞对多种压力的反应中起着关键作用。HF也是一个真正的毒力因子，并导致多药耐药。作为其功能的一部分，Hfq与富含A/U的序列结合，以促进小RNA(SRNAs)与靶mRNAs的退火，通常抑制它们的翻译。因此，Hfq是一种RNA伴侣。Hfq还改变了sRNA和大肠杆菌(EC)的稳定性。对Hfq功能的充分理解在很大程度上是由于缺乏与之相关的Hfq-RNA复合体的高分辨率结构。尽管已报道的HfQ与较小的寡核苷酸结合的结构揭示了HfQ使用两个面结合RNA，但它们留下了许多机制和功能问题没有得到解答。在类核中也发现了Hfq，并与DNA结合。Hfq-DNA的结合机制是一个谜，需要结构研究来解开它。也许剩下的两个关键问题是：HfQ如何与更大的mRNA靶标、sRNA和sRNA-mRNA复合体结合，以及HfQ是否有简单或甚至复杂的序列或基于结构的密码来结合DNA(甚至是RNA)？这些问题的答案对于全面理解Hfq的功能至关重要。因此，考虑到这些大问题，这项高风险、高回报的R21赠款提案有两个具体目标，主要使用结晶学。第一个具体目的是确定与生理相关的同源sRNAs、mRNAs及其三元复合体结合的革兰氏阴性和革兰氏阳性细菌的HfQ的结构。作为这一目标的一个组成部分，色氨酸荧光猝灭实验使用了一系列来自大肠杆菌和单核细胞增多性乳杆菌的含有色氨酸的Hfq蛋白，将提供每个蛋白质上RNA结合位点的初始、指导的较低分辨率图。第二个具体目的是利用固有弯曲的dsDNA序列、其他dsDNA序列和单链A-链DNA来表征EC Hfq-DNA复合体的生化和结构特征，并使用REPSA和CHAP-SEQ鉴定染色体编码的Hfq-DNA结合位点。这项研究的长期目标是描绘HfQ作为转录后调节因子以及可能作为核类相关转录调节因子发挥作用的结构和生化机制。由于Hfq的缺失可以减弱细菌的毒力，这种蛋白是化疗干预的潜在靶点，所提出的具有生物相关性的Hfq-RNA复合体的结构将为未来的任何药物设计工作提供宝贵的指导。

DESCRIPTION (provided by applicant): Hfq is a pleiotropic, posttranscriptional regulator found in many bacteria. Hfq plays a critical role in the cellular response to multiple stresses. Hf is also a bona fide virulence factor and contributes to multidrug resistance. As part of its function, Hfq binds A/U-rich sequences to facilitate the annealing of small RNAs (sRNAs) to target mRNAs, typically repressing their translation. Hence Hfq is an RNA-chaperone. Hfq also alters the stability of sRNAs and Escherichia coli (Ec). A full understanding of Hfq function has been hampered in great part by the dearth of high-resolution structures of germane Hfq-RNA complexes. Although the reported structures of Hfq bound to smaller oligoribonucleotides reveal that Hfq uses two faces to bind RNA, they leave multiple mechanistic and functional questions unanswered. Hfq is also found in the nucleoid and binds DNA. The Hfq- DNA binding mechanism is a mystery and structural studies are needed to unravel it. Perhaps the two key questions remaining are: how does Hfq bind to larger, mRNA targets, sRNAs and sRNA-mRNA complexes and is there a simple or even complex sequence or structure-based code that Hfq uses to bind DNA (or even RNA)? The answers to these questions are crucial to a complete understanding of Hfq function. Hence with these big questions in mind, this high risk-high reward R21 grant proposal has two Specific Aims that employ primarily crystallography. The first Specific Aim is to determine the structures of Hfq from Gram-negative and Gram-positive bacteria bound to physiologically relevant cognate sRNAs, mRNAs and their ternary complexes. As a component of this Aim, tryptophan fluorescence quenching experiments using a series of single tryptophan-containing Hfq proteins from E. coli and L. monocytogenes will provide an initial, guiding lower- resolution map of the RNA binding sites on each protein. The second Specific Aim is to characterize biochemically and structurally Ec Hfq-DNA complexes using intrinsically curved dsDNA sequences, other dsDNA sequences, and single stranded A-tract DNA and to identify chromosomally encoded Hfq-DNA binding sites using REPSA and ChAP-seq. The longer-term goal of this research is to delineate the structural and biochemical mechanisms that Hfq uses to function as a posttranscriptional regulator and possibly as a nucleoid-associated transcriptional regulator. Since the loss of Hfq attenuates bacterial virulence, this protein is a potential target for chemotherapeutic intervention, and the proposed structures of biologically relevant Hfq- RNA complexes will provide invaluable guidance in any future drug design efforts.