Single Molecule Measurements Of Transcript Elongation And Termination By Rna Polymerase

Author : Manchuta Dangkulwanich
Publisher :
ISBN 13 :
Total Pages : 137 pages
Book Rating : 4.:/5 (928 download)

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Download or read book Molecular Mechanisms of Factors that Control RNA Polymerase II Transcription Elongation Dynamics written by Manchuta Dangkulwanich and published by . This book was released on 2015 with total page 137 pages. Available in PDF, EPUB and Kindle. Book excerpt: The expression of a gene begins by transcribing a target region on the DNA to form a molecule of messenger RNA. As transcription is the first step of gene expression, it is there- fore highly regulated. The regulation of transcription is essential in fundamental biological processes, such as cell growth, development and differentiation. The process is carried out by an enzyme, RNA polymerase, which catalyzes the addition of a nucleotide complementary to the template and moves along the DNA one base pair at a time. To complete its tasks, the enzyme functions as a complex molecular machine, possessing various evolutionarily designed parts. In eukaryotes, RNA polymerase has to transcribe through DNA wrapped around histone proteins forming nucleosomes. These structures represent physical barriers to the transcribing enzyme. In chapter 2, we investigated how each nucleosomal component--the histone tails, the specific histone-DNA contacts, and the DNA sequence--contributes to the strength of the barrier. Removal of the tails favors progression of RNA polymerase II into the entry region of the nucleosome by locally increasing the wrapping-unwrapping rates of the DNA around histones. In contrast, point mutations that affect histone-DNA contacts at the dyad abolish the barrier to transcription in the central region by decreasing the local wrapping rate. Moreover, we showed that the nucleosome amplifies sequence-dependent transcriptional pausing, an effect mediated through the structure of the nascent RNA. Each of these nucleosomal elements controls transcription elongation by distinctly affecting the density and duration of polymerase pauses, thus providing multiple and alternative mechanisms for control of gene expression by additional factors. During transcription elongation, RNA polymerase has been assumed to attain equilibrium between pre- and post-translocated states rapidly relative to the subsequent catalysis. Under this assumption, a branched Brownian ratchet mechanism that necessitates a putative secondary nucleotide binding site on the enzyme was proposed. In chapter 3, we challenged individual yeast RNA polymerase II (Pol II) with a nucleosome as a "road block", and separately measured the forward and reverse translocation rates with our single-molecule transcription elongation assay. Surprisingly, we found that the forward translocation rate is comparable to the catalysis rate. This finding reveals a linear, non-branched ratchet mech-anism for the nucleotide addition cycle in which translocation is one of the rate-limiting steps. We further determined all the major on- and off-pathway kinetic parameters in the elongation cycle. This kinetic model provides a framework to study the influence of various factors on transcription dynamics. To further dissect the operation of Pol II, we focused on the trigger loop, a mobile element near the active site of the enzyme. Biochemical and structural studies have demonstrated that the trigger loop makes direct contacts with substrates and promotes nucleotide incorporation. It is also an important regulatory element for transcription fidelity. In chapter 4, we characterized the dynamics of a trigger loop mutant RNA polymerase to elucidate the roles of this element in transcription regulation, and applied the above kinetic framework to quantify the effects of the mutation. In comparison to the wild-type enzyme, we found that the mutant is more sensitive to force, faster at substrate sequestration, and more efficient to return from a pause to active transcription. This work highlighted important roles of regulatory elements in controlling transcription dynamics and fidelity. Moreover, RNA polymerase interacts with various additional factors, which add layers of regulation on transcription. Transcription factors IIS (TFIIS) and IIF (TFIIF) are known to interact with elongating RNA polymerase directly and stimulate transcription. In chapter 5, we studied the effects of these factors on elongation dynamics using our single molecule assay. We found that both TFIIS and TFIIF enhance the overall transcription elongation by reducing the lifetime of transcriptional pauses and that TFIIF also decreases the probability of pause entry. Furthermore, we observed that both factors enhance the efficiency of nucleosomal transcription. Our findings helped elucidate the molecular mechanisms of gene expression modulation by transcription factors. In summary, we have dissected the mechanisms by which the nucleosomal elements regulate transcription, and derived a quantitative kinetic model of transcription elongation in a linear Brownian ratchet scheme with the slow translocation of the enzyme. The corresponding translocation energy landscape shows that the off-pathway states are favored thermodynamically but not kinetically over the on-pathway states. This observation confers the enzyme its high propensity to pause, thus allowing additional regulatory mechanisms during pausing. TFIIS and TFIIF, for example, regulate transcription dynamics by shortening the lifetime of Pol II pauses. On the other hand, the trigger loop of Pol II regulates both the active elongation and pausing. These examples illustrate molecular mechanisms of cis- and trans-acting factors regulate the dynamics of transcription elongation.