Defining the structural mechanisms of Hfq binding to cognate nucleic acids

描述（由申请人提供）：Hfq是一种多效性转录后调节剂，存在于许多细菌中。Hfq在细胞对多种应激的反应中起着关键作用。Hf也是一种真正的毒力因子，有助于多药耐药。作为其功能的一部分，Hfq结合富含A/ u的序列，促进小rna （sRNAs）退火到靶mrna，通常抑制它们的翻译。因此Hfq是一种rna伴侣。Hfq还能改变sRNAs和大肠杆菌（Ec）的稳定性。由于缺乏相关Hfq- rna复合物的高分辨率结构，对Hfq功能的充分了解在很大程度上受到了阻碍。虽然报道的Hfq与较小的寡核苷酸结合的结构表明Hfq使用两个面来结合RNA，但它们留下了多个机制和功能问题未解之谜。Hfq也存在于类核中并与DNA结合。Hfq- DNA的结合机制是一个谜，需要通过结构研究来解开它。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复合物结合，以及Hfq用于结合DNA（甚至RNA）是否存在简单或复杂的序列或基于结构的代码？这些问题的答案对于全面理解Hfq功能至关重要。因此，考虑到这些大问题，这个高风险、高回报的R21拨款提案有两个主要使用晶体学的具体目标。第一个特定目的是确定与生理相关的同源sRNAs、mrna及其三元复合物结合的革兰氏阴性和革兰氏阳性细菌的Hfq结构。作为该Aim的一个组成部分，使用一系列来自大肠杆菌和单核细胞生长乳杆菌的含有单个色氨酸的Hfq蛋白进行色氨酸荧光猝灭实验，将提供每个蛋白上RNA结合位点的初始、可指导的低分辨率图谱。第二个特定目标是使用内在弯曲的dsDNA序列、其他dsDNA序列和单链a链DNA来表征Ec Hfq-DNA复合物的生化和结构特征，并使用REPSA和chapp -seq鉴定染色体编码的Hfq-DNA结合位点。本研究的长期目标是描述Hfq作为转录后调节剂和可能作为核相关转录调节剂的结构和生化机制。由于Hfq的缺失会减弱细菌的毒力，因此该蛋白是化疗干预的潜在靶点，并且提出的生物学相关Hfq- RNA复合物的结构将为任何未来的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.