Transcription Elongation Control in E. coli

描述（由申请人提供）：转录延伸是RNA聚合酶（RNAP）将DNA复制成RNA的过程，是所有生物基因调控的关键点。延伸不是一个单调的过程，可以被暂停、逮捕、路障和终止信号打断。这些事件受到控制RNA产物形成速率的各种因素的调节。这项工作的长期目标是确定维持活细菌细胞中转录的适当速率、进程和准确性的基本机制。本建议的具体目的是：1。大肠杆菌中控制转录速率和转录率的机制的阐明。初步研究表明，结构和稳定RNA基因的转录延伸率取决于生长速度和各种胁迫条件，并受到新机制的调控。我们建议通过实验来详细描述这些机制，并了解它们在细胞适应环境变化中的作用。2. 对控制？在大肠杆菌中循环最近的研究表明，来自大肠杆菌的RNAP群体保留了起始特异性？因素(?70)全程延伸。该种群的相对数量取决于细胞的生长情况，在静止期达到最大值。此外，？-保留群体在某些启动子上支持多轮转录的能力要高得多，这表明它在基因调控中起着重要作用。提出了实验来确定分子机制，控制释放？70和其他a因素。3. 大肠杆菌转录伸长校对机制的阐明。在体内，大肠杆菌RNAP的错误率估计为~ 1 /1.2x105。然而，如此高的准确度的决定因素是未知的。初步数据表明，大肠杆菌中存在一种未知因子，可激活RNAP固有的3‘-5’外切酶（校对）活性。我们建议在体外分离和表征该因子，并确定其在维持体内转录保真度中的作用。我们还将进一步研究GreA/GreB转录物切割因子是否对体内转录保真度有贡献。

DESCRIPTION (provided by applicant): Transcription elongation, the process of copying DNA into RNA by the RNA polymerase (RNAP), is a crucial point of gene regulation in all organisms. Elongation is not a monotonic process and can be interrupted by pauses, arrests, roadblocks, and termination signals. These events are subject to modulation by various factors that control the rate of RNA product formation. The long-term objective of the proposed work is to determine the basic mechanisms that maintain the proper rate, processivity, and accuracy of transcription in living bacteria cells. Specific aims of this proposal are: 1. Elucidation of the mechanisms that control the rate and processivity of transcription in E. coli. Preliminary studies demonstrate that the rate of transcription elongation of structural and stable RNA genes depends on growth rate and various stress conditions and is regulated by novel mechanisms. Experiments are proposed to characterize these mechanisms in detail and understand their role in cell adaptation to environmental changes. 2. Elucidation of the mechanism that controls the ? cycle in E. coli. Recent studies indicate that a population of RNAP from E. coli retains initiation-specific ? factor (?70) throughout elongation. The relative amount of this population depends on cellular growth and reaches its maximum during stationary phase. Moreover, the ?-retaining population has a substantially higher ability to support multiple rounds of transcription at certain promoters, suggesting its significant role in gene regulation. Experiments are proposed to determine the molecular mechanism, that controls the release of ?70 and other a factors during elongation. 3. Elucidation of the proofreading mechanism of transcription elongation in E. coli. In vivo, error rates for E. coli RNAP have been estimated to be ~l/1.2x105. However, the determinants of such a high accuracy are unknown. Preliminary data indicates that an unidentified factor exists in E. coli that activates the intrinsic 3'-5' exonuclease (proofreading) activity of RNAP. Experiments are proposed to isolate and characterize this factor in vitro and determine its role in maintaining transcription fidelity in vivo. It is further proposed to examine whether GreA/GreB transcript cleavage factors contribute to transcription fidelity in vivo.