The Effect of DNA Bending on Transcriptional Elongation by E. coli RNA Polymerase

描述（由申请人提供）：该项目的总体目标是阐明DNA中局部结构扰动对RNA聚合酶（RNAP）转录DNA形成RNA产物的影响。转录是DNA中遗传信息表达的第一步，因此其调控是基因表达调控的核心。在RNA链延伸过程中，由RNAP、其DNA模板和新生RNA产物组成的三元复合物的顺利和快速进展可能受到DNA模板或RNA产物的各种序列和/或结构特征的内在阻碍，并受到与RNAP相关的各种外在因素的调节。延长调控机制的缺陷与许多人类疾病有关，因此参与这一过程的因素是重要的预防和治疗靶点。为了理解转录延伸的调控，我们必须首先了解RNAP与其DNA模板和控制延伸的RNA产物之间的相互作用。很明显，RNAP与下游DNA之间的相互作用可以在转录复合物沿DNA的各个方面发挥重要作用，从启动子逃逸到暂停和终止。然而，这些作用背后的机制是未知的，正如外源调节蛋白影响这些作用的方式一样。对下游DNA序列对延伸的影响的观察（包括我自己的初步研究）包括暂停和阻止上游a束或t束的例子，据推测，这些影响可能是由于a束（或t束）诱导的DNA弯曲。目前还没有对这种下游DNA结构对转录延伸的影响进行系统的研究。我建议使用一系列包含系统变化的弯曲位点的转录模板来探索大肠杆菌RNAP转录中DNA弯曲和RNA链延伸动力学之间的关系。主要使用体外生化方法采用这些模板，突变形式的RNAP已知在其延伸特性改变，以及外部调节蛋白已知影响延伸过程，我计划阐明的机制(s) DNA弯曲影响延伸。

DESCRIPTION (provided by applicant): The overall goal of this project is to elucidate the effects that local structural perturbations in DNA have on an advancing RNA polymerase (RNAP) as it transcribes DNA to form an RNA product. Transcription is the first step in the expression of genetic information in DNA, and as such its regulation is central to the regulation of gene expression. During RNA chain elongation, smooth and rapid progress of the ternary complex, consisting of RNAP, its DNA template, and nascent RNA product, can be hampered intrinsically by a variety of sequence and/or structural features of the DNA template or RNA product, and regulated by a variety of extrinsic factors that associate with RNAP. Defects in elongation regulatory mechanisms have been associated with many human diseases, and therefore the factors involved in this process are important preventive and therapeutic targets. To understand the regulation of transcriptional elongation, we must first understand the interactions between RNAP and its DNA template and RNA product that govern elongation. It is clear that interactions between RNAP and downstream DNA can play significant roles in all aspects of progress of the transcription complex along the DNA, from promoter escape to pausing and termination. However, the mechanism(s) underlying these roles are unknown, as are the ways in which extrinsic regulatory proteins affect these roles. Observations (including my own preliminary studies) of effects of downstream DNA sequences on elongation have included examples of pausing and arrest upstream of A-tracts or T-tracts, and it has been speculated that these effects may be due to A-tract- (or T-tract-) induced DNA bending. No systematic study of the effects of such downstream DNA structures on transcriptional elongation has been undertaken. I propose to use a series of transcription templates containing a systematically varied site of bending to explore the relationship between DNA bending and RNA chain elongation kinetics in transcription by E. coli RNAP. Using primarily an in vitro biochemical approach employing these templates, mutant forms of RNAP known to be altered in their elongation properties, as well as extrinsic regulatory proteins known to affect the elongation process, I plan to elucidate the mechanism(s) by which DNA bending affects elongation.