Regulation of transcription elongation

转录延伸的调控

• 批准号: 8370811
• 负责人: PAUL L BABITZKE
• 金额: $ 27.55万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8370811
• 关键词: Address; Affect; Amino Acids; Bacillus subtilis; Bacteria; Bacterial Genome; Biochemical; Biological Models; Cell physiology; Code; Complex; DNA; DNA-Directed RNA Polymerase; Elongation Factor; Escherichia coli; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genetic; Genetic Transcription; Genome; Genomics; HIV; Health; Homologous Gene; Human; In Vitro; Life; Malignant Neoplasms; Nucleic Acids; Nucleotides; Oncogenes; Operon; Organism; Physiological; Play; Positioning Attribute; Prevalence; Proteins; RNA; Regulation; Repression; Role; Signal Transduction; Site; Site-Directed Mutagenesis; Stretching; Structural Models; Structure; Testing; Time; Transcription Elongation; Translations; attenuation; crosslink; human disease; improved; in vivo; insight; interest; mutant; novel; protein function; research study; stem; transcription factor; transcription termination

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. PUBLIC HEALTH RELEVANCE: Insight into transcription elongation mechanisms will be sought through studies of RNAP pausing and transcription termination in Bacillus subtilis and Escherichia coli. Proper regulation of gene expression is of paramount importance for the function of cells in all organisms. As several human diseases such as cancer arise in part through inappropriate gene expression, while expression of HIV and certain oncogenes are regulated by attenuation, these studies will contribute to improving human health by providing a detailed mechanistic understanding of how transcription elongation is controlled.

描述（由申请人提供）：控制转录伸长和终止的机制是所有生物体中基因表达的重要组成部分。我们开发了枯草芽孢杆菌TRP操纵子领导者作为RNA聚合酶（RNAP）暂停和内在终止的模型系统。我们确定了B. uttilis TRP领导者（U107和U144）中的两个暂停站点， 分别参与转录衰减和翻译抑制机制，尽管仅为体内的翻译抑制机制建立了这一点。 NUSA和NUSG在两个地点都刺激了暂停。尽管已知NUSA刺激大肠杆菌中的暂停，但NUSG刺激的暂停与鉴定出的抗PA的活性相反。 NUSG（SPT5）是唯一普遍保守的转录因子。由于我们确定了任何生物体中NUSG/SPT5刺激的暂停的唯一示例，因此我们处于独特的位置，可以研究这种新型的NUSG功能。我们的结果表明，NUSG与暂停的转录气泡中的非板板DNA链进行了序列特异性接触。我们将确定暂停复合物中NUSG和核酸之间的特定接触。我们还将测试通过位置诱变的NUSG刺激的结构模型。此外，我们将使用基因组方法来识别整个枯草芽孢杆菌基因组中NUSG刺激的暂停位点，以确定这种暂停机制的患病率，这可能在生命的所有三个领域中都保留。 虽然假定RNAP暂停在几种衰减机制中起作用，但在体内的任何衰减机制中从未显示出这种情况。使用我们以前的U144暂停研究作为指导，我们将产生暂停缺陷的突变体，这将使我们能够确定在U107处暂停是否参与体内枯草芽孢杆菌TRP操纵子的衰减机制。 规范的固有终结器由不间断的RNA发夹组成，然后是usectues。多年来，NUSA能够刺激内在终止者约10-20％的终止。特别有趣的是，我们发现来自枯草芽孢杆菌和大肠杆菌的NUSA可以大大提高含有发夹不匹配和/或较差的U区（最多18倍）的弱非规范终止剂的终止效率。尽管终止并不严格依赖于NUSA，但我们将此机制称为NUSA依赖性终止，将其与规范终止剂处发生的轻微刺激区分开。由于已经在各种细菌物种中预测了数百种非典型终止剂，因此终结剂的数量似乎远高于以前想象的。我们将检查枯草芽孢杆菌和大肠杆菌中的NUSA依赖性终止，以确定这种先前被忽视的终止机制是否在细菌中保守。最后，我们发现NUSA依赖性终止可以调节体外的转录读取到NUSA编码序列中。我们将进一步表征这种新型的自动调节衰减机制，该机制似乎依赖于NUSA依赖性终止而不是重叠的RNA结构。 公共卫生相关性：通过研究枯草芽孢杆菌和大肠杆菌中的RNAP暂停和转录终止，将寻求有关转录伸长机制的见解。适当的基因表达调节对于所有生物体中细胞的功能至关重要。由于几种人类疾病（例如癌症）部分通过不适当的基因表达而产生，而艾滋病毒和某些癌基因的表达受到衰减的调节，这些研究将通过对转录伸长的控制方式提供详细的机械理解，从而有助于改善人类健康。