Mechanims of Archaeal Transcription Termination

描述（由申请人提供）：基因表达的及时和准确调控是正常生长、发育和对环境刺激的反应所必需的。完全理解用于调节基因表达的机制对于对抗作为许多人类发育条件和疾病（包括癌症）的基础的异常调节是必要的。该提案的长期目标是确定DNA依赖性多亚基RNA聚合酶（RNAP）的RNA合成机制，保守蛋白质因子对RNAP的调节和选择模板序列，并具体描述停止RNA合成和终止转录的可用机制。所提出的实验利用体内和体外建立的古细菌转录系统。在人类分子生物学的许多特征中，特别是RNAP结构和功能的保守性方面，RNP的复杂性要低得多，但具有同源性。我们将选择，产生，纯化和表征变异RNAP与修改转录终止表型。我们将进一步表征因子依赖性终止，极性，以及这种调节在Escherichia中的作用。如此获得的结果将确定支持转录延伸复合物稳定性的结构和序列，描述终止的机制，并允许在每个域中的终止机制的比较，并打开一个新的研究领域，古细菌因子依赖性终止。

DESCRIPTION (provided by applicant): Timely and accurate regulation of gene expression is required for proper growth, development, and response to environmental stimuli. A complete understanding of the mechanisms employed to regulate gene expression is necessary to combat the aberrant regulation that underlies many human developmental conditions and ailments, including cancer. The long-term goals of this proposal are to determine the mechanics of RNA synthesis by DNA-dependent multi-subunit RNA polymerases (RNAPs), the regulation imposed on RNAP by conserved protein factors and select template sequences, and to specifically characterize the available mechanisms to halt RNA synthesis and terminate transcription. The proposed experiments take advantage of both an in vivo and in vitro established archaeal transcription system. Archaea offer the advantages of far less complexity but homology in many features of human molecular biology, specifically conservation of RNAP structure and function. We will select, generate, purify and characterize variant RNAPs with modified transcription termination phenotypes. We will further characterize factor-dependent termination, polarity, and the role of such regulation in Archaea. The results so obtained will determine the structures and sequences that support transcription elongation complex stability, describe the mechanics of termination and allow comparisons of termination mechanisms in each Domain, and open a new field of study, archaeal factor-dependent termination.