Processive Antitermination of Antibiotic Synthesis Genes

摘要 NusG是一种转录延伸蛋白，几乎被来自生命三个领域的所有生物使用。细菌 NusG通过其N-末端结构域与RNA聚合酶(RNAP)结合，而尽管它的尺寸很小，但 C末端结构域(CTD)与其他转录因子(Rho、S10、NusB和 Nusa)影响转录延伸。虽然所有的细菌都编码核心的NusG，但许多细菌也合成了 与RNAP瞬时结合以改变目标基因表达的Paralog。然而，尽管 他们调控的基因，大多数已知的NusG近亲动物亚家族还没有被深入研究(例如， UpxY、TAA和ACTx)。我们最近发现了一个新的、分布广泛的NusG样蛋白亚家族，它 我们给LoaP打电话了。我们对这种独特的蛋白质的初步研究表明，绒毛芽孢杆菌LoaP 激活由两个不同的抗生素合成操纵子组成的调节子的表达。在进一步 检查后，我们发现有证据表明LoaP和抗生素之间存在广泛的调控关系 合成操纵子。这一发现特别重要，因为我们的数据表明，LoaP监管机构 蛋白质将转录延长机制重新配置为抗终止复合体，能够 绕过遍及整个抗生素合成操纵子的多个终止位点。这些东西的存在 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 如果没有专门的抗终止复合体，转录是不可能的。我们推测这是 观察解释了为什么一些抗生素合成操纵子不能在 异源宿主；它们可能只是积累了显著抑制转录延伸的终止位点 在没有他们的同源抗终止因子的情况下。总而言之，这些数据表明，有一个紧急的 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 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，非常重要的一点是了解 LoaP抗终止机制对这些重要的专门化代谢产物产生调节作用 歌剧。此外，我们的初步数据已经表明，LoaP使用一种监管机制，即 不同于其他已知的NusG Paralog使用的那些。在这个项目中，我们将发现分子 LoaP用来专门操作抗生素合成操纵子转录延伸的机制。这 将大大扩展控制抗生素合成的调节机制的知识，同时还 揭示了转录延伸复合体的工作原理的新的和基本的见解。

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.