Conformational Dynamics of T7 RNAP Transcription

T7 RNAP是广泛存在的一类RNAP中研究最充分的成员，所述RNAP包括噬菌体编码的RNAP、质体和质粒编码的酶以及叶绿体和线粒体RNAP。它还显示出与DNAP、RT和RNA指导的RNAP更有限的序列相似性，但具有广泛的结构相似性。 T7 RNAP在T7生命周期中扮演着多种角色。除了转录噬菌体基因外，它还引发T7 DNA复制，并可能在T7串联体连接处启动T7 DNA包装和成熟机制的组装。相对简单的单亚基T7 RNAP似乎能够进行这些复杂的反应-通常由更大的多亚基酶执行-因为它具有非凡的构象适应性。由于其相对简单的结构，机制的复杂性，以及我们对这种分子的理解的先进状态，T7 RNAP提供了一个非常有吸引力的系统，在其中追求分子生物学的中心目标：理解机制 进行细胞过程的细胞。在下一个项目期间，我们将处理这个系统中尚未解决或得到相互矛盾的答案的悬而未决的问题。具体来说，我们将描述的结构转变所经历的转录复合物在初始转录的后期阶段和过渡到成熟EC的不成熟。我们将询问如何在启动子释放点处产生破坏启动子：RNAP相互作用的力，并表征单个氨基酸在延伸过程中易位的作用。构象变化期间暂停和终止在T7串联体连接和T7 Tphi（发夹）终止子将进行研究的方法，允许时间分辨的瞬态复合物的表征。最后，T7 RNAP引发T7 DNAP的特征在于回答以下问题：引物如何从RNAP转移到DNAP，以及主要来源的序列如何有助于这一过程？

T7RNAP is the most well studied member of a widespread class of RNAPs that includes phage-encoded RNAPs, plastid and plasmid encoded enzymes, and chloroplast and mitochondrial RNAPs. It also displays more limited sequence similarity, but extensive structural similarity, to DNAPs, RTs & RNA-directed RNAPs. T7RNAP plays multiple roles in the T7 life cycle. In addition to transcribing the phage genes, it primes T7 DNA replication and probably acts to initiate assembly of the T7DNA packing and maturation machinery at the T7 concatemer junction. The relatively simple single-subunit T7RNAP appears able to carry out these complex reactions--usually executed by much larger multi-subunit enzymes--because it of its extraordinary conformational adaptability. Because of its relative structural simplicity, mechanistic complexity, and the advanced state of our understanding of this molecule, T7RNAP presents an exceptionally attractive system in which to pursue a central ambition of molecular biology: understanding the mechanisms of the machinery that carries out cellular processes. Over the next project period we will address the outstanding questions which have yet to be tackled, or have received conflicting answers, in this system. Specifically, we will characterize the structural transitions undergone by the transcription complex during the late stages of initial transcription and the transition from the immature to the mature EC. We will ask how the force that ruptures the promoter:RNAP interaction at the point of promoter release is generated, and characterize the role of individual amino acids in translocation during elongation. Conformational changes during pausing and termination at the T7 concatemer junction and the T7 Tphi (hairpin) terminator will be studied with approaches that allow time-resolved characterization of transient complexes. Finally, T7RNAP priming of T7 DNAP will be characterized with the goal of answering the following questions: how is the primer transferred from RNAP to DNAP and how does the sequence of the primary origin contribute to this process?