Defining the structural mechanisms of Hfq binding to cognate nucleic acids

描述（由申请人提供）：Hfq是一种在许多细菌中发现的多效性转录后调节因子。Hfq在细胞对多种应激的反应中起关键作用。铪也是一种真正的毒力因子，并有助于多药耐药性。作为其功能的一部分，Hfq结合富含A/U的序列以促进小RNA（sRNA）与靶mRNA的退火，通常抑制它们的翻译。因此Hfq是RNA分子伴侣。Hfq还改变sRNA和大肠杆菌（Ec）的稳定性。对Hfq功能的全面了解在很大程度上受到缺乏密切相关的Hfq-RNA复合物的高分辨率结构的阻碍。尽管报道的Hfq与较小的寡核苷酸结合的结构揭示了Hfq使用两个面来结合RNA，但它们留下了许多机制和功能问题没有答案。Hfq也存在于类核中并结合DNA。Hfq- DNA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靶点、sRNA和sRNA-mRNA复合物结合，以及Hfq是否使用简单甚至复杂的序列或基于结构的密码来结合DNA（甚至RNA）？这些问题的答案对于完全理解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采用晶体学的具体目标。第一个具体目的是确定与生理学相关的同源sRNA、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的结构。作为这一目标的组成部分，色氨酸荧光猝灭实验使用了一系列单一的含色氨酸的Hfq蛋白从E。coli和L.单核细胞增多症将提供每个蛋白质上RNA结合位点的初始的、指导性的低分辨率图谱。第二个具体目标是使用固有弯曲的dsDNA序列、其他dsDNA序列和单链A段DNA表征Ec Hfq-DNA复合物的生物化学和结构，并使用REPSA和ChAP-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作为转录后调节因子和可能作为类核相关转录调节因子发挥作用的结构和生化机制。由于Hfq的缺失减弱了细菌的毒力，因此该蛋白质是化学治疗干预的潜在靶标，并且所提出的生物学相关Hfq-RNA复合物的结构将在任何未来的药物设计工作中提供宝贵的指导。

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.