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.

抽象的 NUSG是几乎所有生物领域的生物使用的转录伸长蛋白。细菌 NUSG通过其N末端结构域与RNA聚合酶（RNAP）相关联，而dospite的大小很小， C末端结构域（CTD）与其他转录因子形成动态相互作用（Rho，S10，NUSB和 NUSA）影响转录伸长。虽然所有广播编码为核心NUSG，但许多人也合成 临时结合RNAP以改变靶向基因的表达的旁系同源物。然而，dospite的重要性 它们调节的基因，尚未深入研究NUSG旁系同源物的大多数已知亚家族（例如，例如， UPXY，TAA和ACTX）。我们最近发现了一种新的和宽度的亚科NUSG样蛋白质，它 我们打电话给一项。我们对这种独特蛋白的初步研究表明，velezensis Loap 激活由两个不同的抗生素合成操纵子组成的常规表达。进一步 检查，我们发现证据表明，提示面和抗生素之间存在广泛的调节关系 合成操纵子。这一发现尤其重要，因为我们的数据表明该产品调节性 蛋白质将转录伸长机械重新配置为抗授予复合物，能够 绕过多个终止位点遍布整个抗生素合成操纵子。这些的存在 终止站点表明，这些操纵子已经“上瘾”对起伏的抗授权因素，因为他们 如果没有专用的抗通过复合物，转录将是不可能的。我们推测这个 观察解释了为什么某些抗生素合成操纵子在一个范围内表达不好 异源宿主；他们可能简单地积累了终止位点，可以抑制转录伸长率 在缺乏同源抗性因子的情况下。这些数据一起证明了如何紧急 需要更好地了解影响抗生素合成操纵子的遗传调节机制，如 信息将影响发现新型抗生素的策略，并将为新工具提供新的工具 改善抗生素的异源产生。因此，了解如何了解 采用抗释放机制对这些重要的专业代谢产生了调节作用 操纵子。此外，我们的初步数据表明，LOAP使用一种调节机制 与其他已知的NUSG旁系同源物使用的不同。在这个项目中，我们将发现分子 LAP用来专门操纵抗生素合成操纵子的转录伸长的机制。这 将显着扩大控制抗生素合成的调节机制的知识 揭示了对转录伸长综合体的工作的新的和基本的见解。