Transcription Elongation Control in E. coli

描述（由申请人提供）：转录延伸，即通过RNA聚合酶（RNAP）将DNA复制成RNA的过程，是所有生物体中基因调控的关键点。伸长不是一个单调的过程，可以被暂停、停止、障碍和终止信号打断。这些事件受到控制RNA产物形成速率的各种因素的调节。拟议工作的长期目标是确定在活细菌细胞中维持适当转录速率、持续合成能力和准确性的基本机制。本申请的具体目的是： 1.阐明了控制E.杆菌初步研究表明，结构和稳定的RNA基因的转录延长的速度取决于生长速度和各种压力条件，并受到新的机制。实验提出详细描述这些机制，并了解它们在细胞适应环境变化中的作用。 2.阐明了E.杆菌最近的研究表明，来自E.大肠杆菌在整个延伸过程中保留起始特异性S因子（S 70）。该群体的相对量取决于细胞生长，并在稳定期达到最大值。此外，保留s的群体具有显著更高的能力来支持在某些启动子处的多轮转录，这表明其在基因调控中的深远作用。实验提出了确定的分子机制，它控制的S 70和替代的S因子在伸长过程中的释放。 3.阐明了E.杆菌在体内，E. coli RNAP的平均值为-1/1.2x105。然而，这种高精度的决定因素是未知的。初步数据表明，在E.大肠杆菌中，激活RNAP内在的3 '-5'核酸外切酶（校对）活性。实验提出了在体外分离和表征这个因子，并确定其在维持体内转录保真度的作用。进一步提出检查GreA/GreB转录物切割因子是否有助于体内转录保真度。

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 application are: 1. Elucidate 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. Elucidate the mechanism that controls the s cycle in E. coli. Recent studies indicate that a population of RNAP from E. coli retains initiation-specific s factor (s 70) throughout elongation. The relative amount of this population depends on cellular growth and reaches its maximum during stationary phase. Moreover, the s - retaining population has a substantially higher ability to support multiple rounds of transcription at certain promoters, suggesting its profound role in gene regulation. Experiments are proposed to determine the molecular mechanism, which controls the release of s 70 and alternative s factors during elongation. 3. Elucidate the proofreading mechanism of transcription elongation in E. coli. In vivo, error rates for E. coli RNAP have been estimated to be -1/1.2x 105. However, the determinants of such a high accuracy are unknown. The preliminary data indicate 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.