Conformational Dynamics of T7 RNAP Transcription

T7RNAP是一类广泛存在的RNAP中研究最多的成员，RNAP包括噬菌体编码的RNAP、质粒编码的酶以及叶绿体和线粒体的RNAP。与DNAPs、RTS和RNA定向的RNAP相比，它也表现出更有限的序列相似性，但结构上的相似性更大。 T7RNAP在T7生命周期中扮演多种角色。除了转录噬菌体基因外，它还启动T7DNA复制，并可能在T7串联连接处启动T7DNA包装和成熟机制的组装。相对简单的单亚基T7RNAP似乎能够执行这些复杂的反应--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T7RNAP相对结构简单，机制复杂，以及我们对该分子理解的先进状态，T7RNAP呈现了一个特别吸引人的系统，在其中追求分子生物学的核心目标：了解机械的机制 进行细胞过程的生物。在下一个项目期间，我们将处理这一系统中尚未解决或已收到相互矛盾的答案的未决问题。具体地说，我们将描述转录复合体在初始转录的后期阶段以及从不成熟的EC到成熟的EC的结构转变。我们将询问在启动子释放点打破启动子：RNAP相互作用的力是如何产生的，并表征单个氨基酸在伸长过程中的易位中的作用。将用能够对瞬时络合物进行时间分辨表征的方法来研究T7串联蛋白结和T7 Tphi(发夹)终止子暂停和终止过程中的构象变化。最后，T7的T7RNAP启动 DNAP的特点是回答以下问题：引物是如何从RNAP转移到DNAP的，以及初级起源的序列对这一过程有何贡献？

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?