Defining the structural mechanisms of Hfq binding to cognate nucleic acids

定义 Hfq 与同源核酸结合的结构机制

基本信息

批准号：
9094550
负责人：
RICHARD GERALD BRENNAN
金额：
$ 22.73万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-07-01 至 2017-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9094550
关键词：
Antibiotics Applications Grants Attenuated Bacteria Binding Binding Sites Biochemical Biochemical Genetics Categories Cells Code Complex Crystallography DNA DNA Binding DNA Sequence DNA-Binding Proteins Distal Drug Design Elements Escherichia coli Escherichia coli Proteins Face Fluorescence Future Gene Expression Gene Targeting Genetic study Goals Gram-Negative Bacteria Gram-Positive Bacteria Health Integration Host Factors Intervention Laboratories Lead Left Listeria monocytogenes Maps Messenger RNA Mind Molecular Chaperones Multi-Drug Resistance Nucleic Acid Binding Nucleic Acids Oligoribonucleotides Play Poly(A) Tail Post-Translational Regulation Proteins RNA RNA Binding RNA Phages RNA Stability Regulation Regulator Genes Reporting Research Resolution Ribosomes Role Series Signal Transduction Single-Stranded DNA Site Small RNA Stress Stretching Structure Translations Tryptophan Untranslated RNA Untranslated Regions Virulence Virulence Factors Work base ds-DNA environmental stressor high reward high risk in vivo insight mRNA Decay mRNA Transcript Degradation novel pathogen pathogenic bacteria research study response ribonuclease E stem

项目摘要

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.

描述（由申请人提供）：Hfq 是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着至关重要的作用。 Hf 也是一种真正的毒力因子，有助于产生多重耐药性。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) 与目标 mRNA 退火，通常抑制其翻译。因此 Hfq 是一种 RNA 伴侣。 Hfq 还会改变 sRNA 和大肠杆菌 (Ec) 的稳定性。由于缺乏锗烷 Hfq-RNA 复合物的高分辨率结构，对 Hfq 功能的全面理解在很大程度上受到阻碍。尽管报道的 Hfq 与较小的寡核糖核苷酸结合的结构表明 Hfq 使用两个面来结合 RNA，但它们留下了多个未解答的机制和功能问题。 Hfq 也存在于类核中并与 DNA 结合。 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 靶标、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代码来结合 DNA（甚至 RNA）？这些问题的答案对于完整理解 Hfq 功能至关重要。因此，考虑到这些重大问题，这项高风险高回报的 R21 拨款提案有两个主要采用晶体学的具体目标。第一个具体目标是确定与生理相关的同源 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 结构。作为该目标的一个组成部分，使用来自大肠杆菌和单核细胞增生李斯特菌的一系列含单一色氨酸的 Hfq 蛋白进行色氨酸荧光猝灭实验将提供每种蛋白上 RNA 结合位点的初始、指导性较低分辨率图。第二个具体目标是使用本质弯曲的 dsDNA 序列、其他 dsDNA 序列和单链 A-tract DNA 表征 Ec Hfq-DNA 复合物的生化和结构特征，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 用作转录后调节因子并可能作为核素相关转录调节因子的结构和生化机制。由于 Hfq 的丢失会减弱细菌的毒力，因此该蛋白是化疗干预的潜在靶标，并且所提出的生物学相关 Hfq-RNA 复合物的结构将为任何未来的药物设计工作提供宝贵的指导。