Mechanims of Archaeal Transcription Termination

描述(由申请人提供)：对于适当的生长、发育和对环境刺激的反应，需要及时和准确地调节基因表达。对基因表达调控机制的全面了解，对于抗击导致许多人类发育状况和疾病(包括癌症)的异常调控是必要的。该建议的长期目标是确定DNA依赖的多亚单位RNA聚合酶(RNAP)合成RNA的机制，保守的蛋白质因子和选择模板序列对RNAP施加的调节，并具体表征可用于停止RNA合成和终止转录的机制。拟议的实验利用了体内和体外建立的古菌转录系统。古生菌的优点是复杂性低得多，但在人类分子生物学的许多特征上是同源的，特别是RNAP结构和功能的保守。我们将选择、产生、纯化和鉴定具有修饰转录终止表型的变异RNAP。我们将进一步表征依赖因子的终止、极性以及这种调控在古生代中的作用。所获得的结果将确定支持转录延伸复合体稳定性的结构和序列，描述终止的机制并允许比较每个结构域的终止机制，并开辟一个新的研究领域，即古代因子依赖的终止。 与公共健康相关：完全了解用于调节基因表达的机制对于对抗异常调节是必要的，这种异常调节是许多人类发育状况和疾病(包括癌症)的基础。该建议的长期目标是确定DNA依赖的多亚单位RNA聚合酶(RNAP)合成RNA的机制，保守的蛋白质因子和选择模板序列对RNAP施加的调节，并具体表征可用于停止RNA合成和终止转录的机制。所获得的结果将确定支持转录延伸复合体稳定性的结构和序列，描述终止机制并允许比较每个结构域的终止机制，并开辟一个新的研究领域，即古代因子依赖终止。

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. PUBLIC HEALTH RELEVANCE: 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 results so obtained will determine the structures and sequences that support transcription elongation complex stability, describe the mechanisms of termination and allow comparisons of termination mechanisms in each Domain, and open a new field of study, archaeal factor-dependent termination.