Processive Antitermination of Antibiotic Synthesis Genes

抗生素合成基因的持续抗终止

• 批准号: 10581588
• 负责人: Wade Winkler
• 金额: $ 33.88万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-01 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10581588
• 关键词: Affect; Affinity; Anabolism; Antibiotics; Bacillus; Bacteria; Base Sequence; Binding; Biochemical; Bioinformatics; Bypass; C-terminal; Complex; DNA-Directed RNA Polymerase; Data; Dedications; Development; Experimental Genetics; Family; Gene Expression; Genes; Genetic; Genetic Transcription; In Vitro; Investigation; Knowledge; Life; Maps; Molecular; Molecular Genetics; Mutation; N-terminal; Names; Operon; Organism; Pathway interactions; Peptide Initiation Factors; Play; Polysaccharides; Production; Proteins; RNA; RNA Binding; Regulation; Regulon; Resolution; Ribonucleoproteins; Role; Signal Transduction; Site; Structure; Subgroup; Transcription Elongation; Transcription Initiation; Transcriptional Elongation Factors; antitermination; collaborative approach; experimental study; gene synthesis; genetic regulatory protein; improved; in vivo; insight; intermolecular interaction; novel; paralogous gene; protein protein interaction; reconstitution; recruit; rho; termination factor; tool; transcription factor; virtual

Abstract NusG is a transcription elongation protein used by virtually all organisms from the three domains of life. Bacterial NusG associates with RNA polymerase (RNAP) through its N-terminal domain, whilst, despite its small size, the C-terminal domain (CTD) forms dynamical interactions with other transcription factors (Rho, S10, NusB and NusA) to affect transcription elongation. While all bacteria encode for a core NusG, many also synthesize paralogs that transiently bind RNAP to alter expression of targeted genes. Yet, despite the importance of the genes they regulate, most of the known subfamilies of NusG paralogs have not been investigated in depth (e.g., UpxY, TaA, and ActX). We recently discovered a new and widespread subfamily of NusG-like proteins, which we called LoaP. Our preliminary investigation of this unique protein showed that Bacillus velezensis LoaP activates expression of a regulon that is comprised of two different antibiotic synthesis operons. Upon further inspection, we found evidence that suggests a broad regulatory relationship between LoaP and antibiotic synthesis operons. This discovery is particularly important because our data suggests that the LoaP regulatory protein reconfigures the transcription elongation machinery into an antitermination complex, capable of bypassing multiple termination sites spread throughout the antibiotic synthesis operons. The presence of these termination sites suggests that these operons have become ‘addicted’ to the LoaP antitermination factor, as their transcription would be impossible without the dedicated antitermination complex. We speculate that this observation explains why some antibiotic synthesis operons do not express well within the confines of a heterologous host; they may have simply accrued termination sites that strikingly inhibit transcription elongation in the absence of their cognate antitermination factor. Together, these data demonstrate how there is an urgent need to better understand the genetic regulatory mechanisms affecting antibiotic synthesis operons, as this information will influence the strategies used for discovering novel antibiotics and will lead to new tools for improving heterologous production of antibiotics. Therefore, it is of significant importance to understand how the LoaP antitermination mechanism exerts its regulatory influence over these important specialized metabolite operons. Moreover, our preliminary data have demonstrated that LoaP uses a regulatory mechanism that is different than those utilized by the other known NusG paralogs. In this project we will discover the molecular mechanism used by LoaP to specifically manipulate transcription elongation of antibiotic synthesis operons. This will significantly expand knowledge of the regulatory mechanisms that control antibiotic synthesis while also revealing new and fundamental insight into the workings of the transcription elongation complex.

摘要 NusG是一种转录延伸蛋白，几乎所有生物体都使用它。细菌 NusG通过其N-末端结构域与RNA聚合酶（RNAP）结合，而尽管其尺寸小， C-末端结构域（CTD）与其他转录因子（Rho、S10、NusB和NusB）形成动态相互作用。 NusA）以影响转录延伸。虽然所有细菌都编码核心NusG，但许多细菌也合成 瞬时结合RNAP以改变靶基因表达的旁系同源物。然而，尽管重要的是 它们调节的基因，大多数已知的NusG旁系同源物亚家族尚未被深入研究（例如， UpxY、TaA和ActX）。我们最近发现了一个新的广泛存在的NusG样蛋白亚家族， 我们称之为Loap。我们对这种独特蛋白的初步研究表明，Velezensis LoaP 激活由两个不同抗生素合成操纵子组成的调节子的表达。经过进一步 通过检查，我们发现证据表明LoaP和抗生素之间存在广泛的调节关系 合成操纵子。这一发现特别重要，因为我们的数据表明，LoaP调节 蛋白质将转录延伸机制重新配置为抗终止复合物，能够 绕过遍布抗生素合成操纵子的多个终止位点。存在这些 终止位点表明，这些操纵子已经对LoaP抗终止因子“上瘾”，因为它们的 如果没有专用的抗终止复合物，转录将是不可能的。我们推测， 观察解释了为什么一些抗生素合成操纵子不能在一个特定的限制内表达。 异源宿主;它们可能简单地积累了显著抑制转录延伸的终止位点 在不存在其同源抗终止因子的情况下。总之，这些数据表明， 需要更好地了解影响抗生素合成操纵子的遗传调控机制，因为这 信息将影响用于发现新抗生素的策略，并将导致新的工具， 改善抗生素的异源生产。因此，了解如何 LoaP抗终止机制对这些重要的特化代谢物发挥调节作用 操纵子此外，我们的初步数据表明，LoaP使用的调节机制， 与其他已知的NusG旁系同源物所利用的那些不同。在这个项目中，我们将发现 LoaP用于特异性操纵抗生素合成操纵子的转录延伸的机制。这 将大大扩展控制抗生素合成的调节机制的知识， 揭示了对转录延伸复合体工作的新的和基本的见解。