Transcription Elongation Control in E. coli

描述(申请人提供)：转录延伸，即通过RNA聚合酶(RNAP)将DNA复制到RNA中的过程，是所有生物体中基因调控的关键点。延长不是一个单调的过程，可以通过暂停、逮捕、路障和终止信号来中断。这些事件受到控制RNA产物形成速度的各种因素的调节。这项拟议工作的长期目标是确定在活的细菌细胞中保持适当的转录速度、加工能力和准确性的基本机制。本申请的具体目标是： 1.阐明在大肠杆菌中控制转录速度和过程的机制。初步研究表明，结构稳定的RNA基因的转录延伸速率取决于生长速度和各种胁迫条件，并受到新的机制的调节。建议进行实验，以详细描述这些机制，并了解它们在细胞适应环境变化中的作用。 2.阐明在大肠杆菌中控制S循环的机制。最近的研究表明，来自大肠杆菌的RNAP群体在整个伸长过程中保持着起始特异性的S因子(S 70)。这种种群的相对数量取决于细胞的生长，并在稳定期达到最大值。此外，保留S的群体在某些启动子上具有更高的支持多轮转录的能力，这表明它在基因调控中发挥着深远的作用。通过实验确定了在伸长过程中控制S 70释放的分子机制和替代S因子。 3.阐明了大肠杆菌转录延伸的校对机制。在体内，大肠杆菌RNAP的错误率估计为-1/1.2×105。然而，这种高准确度的决定因素是未知的。初步数据表明，在大肠杆菌中存在一种激活RNAP固有的3‘-5’核酸外切酶(校对)活性的未知因子。建议进行实验，在体外分离和鉴定该因子，并确定其在体内维持转录保真度的作用。进一步建议研究GREA/GREB转录切割因子是否有助于体内转录的保真度。

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.