Regulation of transcription elongation

描述(申请人提供)：控制转录延长和终止的机制是所有生物体基因表达的重要组成部分。我们开发了枯草杆菌色氨酸操纵子前导作为RNA聚合酶(RNAP)暂停和内在终止的模型系统。我们在枯草杆菌Trp前导序列中确定了两个停顿位点(U107和U144)， 它们分别参与转录抑制和翻译抑制机制，尽管这只在体内为翻译抑制机制建立。Nusa和NusG在这两个网站上都会刺激暂停。虽然已知NusA能在大肠杆菌中刺激停顿，但NusG刺激的停顿与E.coliNusG所鉴定的抗停顿活性相反。NusG(Spt5)是唯一一个普遍保守的转录因子。由于我们在任何生物体中都发现了NusG/Spt5刺激的暂停的唯一例子，所以我们处于一个独特的位置来研究这一新的NusG功能。我们的结果表明，NusG与暂停转录泡中的非模板DNA链进行了序列特异性接触。我们将确定NusG与暂停的复合体中的核酸之间的特定接触。我们还将通过定点突变来测试NusG刺激的暂停的结构模型。此外，我们将使用基因组学方法在整个枯草杆菌基因组中鉴定NusG刺激的停顿位置，以确定这种停顿机制的流行率，这种停顿机制可能在生命的所有三个领域都是保守的。虽然RNAP暂停被认为在几种衰减机制中起作用，但在活体中从未显示出任何衰减机制。以我们之前的U144暂停研究为指导，我们将产生暂停缺陷突变体，这将使我们能够确定U107处的暂停是否参与了枯草杆菌色氨酸操纵子在体内的衰减机制。典型的内在终止子由一个不间断的RNA发夹和一段U残基组成。多年来，人们已经知道NusA能够刺激在内在终止子~10-20%处终止。特别有趣的是，我们发现来自枯草杆菌和大肠杆菌的NusA可以极大地提高在含有发夹错配和/或差U束的弱非正则末端的终止效率(高达18倍)。虽然终止并不严格依赖于NusA，但我们将这种机制称为NusA依赖终止，以区别于发生在规范终止符的轻微刺激。由于在各种细菌物种中已经预测到数百个非规范终止子，因此终止子的数量似乎比之前认为的要高得多。我们将研究枯草杆菌和大肠杆菌中依赖NusA的终止机制，以确定这种以前被忽视的终止机制是否在细菌中保守。最后，我们发现在体外，NusA依赖的终止调节转录进入NusA编码序列。我们将进一步表征这种新的自动调节衰减机制，它似乎依赖于NusA依赖的终止，而不是重叠的RNA结构。

DESCRIPTION (provided by applicant): Mechanisms that control transcription elongation and termination are important components of gene expression in all organisms. We developed the B. subtilis trp operon leader as a model system for RNA polymerase (RNAP) pausing and intrinsic termination. We identified two pause sites in the B. subtilis trp leader (U107 and U144), which participate in transcription attenuation and translation repression mechanisms, respectively, although this has only been established for the translation repression mechanism in vivo. NusA and NusG stimulate pausing at both sites. Although NusA was known to stimulate pausing in E. coli, NusG- stimulated pausing is opposite to the anti-pausing activity identified fo E. coli NusG. NusG (Spt5) is the only universally conserved transcription factor. Since we identified the only examples of NusG/Spt5-stimulated pausing in any organism, we are in a unique position to investigate this novel NusG function. Our results indicate that NusG makes sequence-specific contacts with the non-template DNA strand within the paused transcription bubble. We will identify specific contacts between NusG and nucleic acids in the paused complex. We will also test a structural model of NusG-stimulated pausing by site-directed mutagenesis. Furthermore, we will use genomic approaches to identify NusG-stimulated pause sites throughout the B. subtilis genome to determine the prevalence of this pausing mechanism, which may be conserved in all three domains of life. While RNAP pausing is assumed to function in several attenuation mechanisms, this has never been shown for any attenuation mechanism in vivo. Using our previous U144 pausing studies as a guide, we will generate pause-defective mutants that will allow us to determine if pausing at U107 participates in the attenuation mechanism of the B. subtilis trp operon in vivo. Canonical intrinsic terminators consist of an uninterrupted RNA hairpin followed by a stretch of U residues. It has been known for many years that NusA is capable of stimulating termination at intrinsic terminators ~10-20%. Of particular interest, we found that NusA from B. subtilis and E. coli can greatly increase the termination efficiency at weak non-canonical terminators containing hairpin mismatches and/or poor U tracts (up to 18-fold). Although termination is not strictly dependent on NusA, we refer to this mechanism as NusA-dependent termination to distinguish it from the slight stimulation that occurs at canonical terminators. As hundreds of non-canonical terminators have been predicted in a variety of bacterial species, it appears that the number of terminators is far higher than previously thought. We will examine NusA-dependent termination in both B. subtilis and E. coli to determine if this previously overlooked termination mechanism is conserved in bacteria. Finally, we found that NusA-dependent termination regulates transcriptional readthrough into the nusA coding sequence in vitro. We will further characterize this novel autoregulatory attenuation mechanism that appears to rely on NusA-dependent termination rather than overlapping RNA structures.