Molecular Characterization of Yeast Factors Involved in Premessenger RNA 3'-end Processing and of Their Function in Transcription Termination by RNA Polymerase II PDF Download
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Author: Martin Sadowski
Publisher:
ISBN:
Category :
Languages : en
Pages : 222
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Author: Martin Sadowski
Publisher:
ISBN:
Category :
Languages : en
Pages : 222
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Author: Sankar Adhya
Publisher: Elsevier
ISBN: 0080522602
Category : Science
Languages : en
Pages : 715
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Book Description
RNA polymerase is molecule important to gene transcription. Along with associated factors, RNA polymerase is part of the process in which RNA is transcribed to produce a protein.* Models and methods for studying polymerase translocation* Assay for movements of RNA polymerase along DNA* Engineering of elongation complexes of bacterial and yeast RNA polymerases
Author:
Publisher:
ISBN:
Category : Medicine
Languages : en
Pages : 1104
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Author: Manchuta Dangkulwanich
Publisher:
ISBN:
Category :
Languages : en
Pages : 137
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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.
Author:
Publisher:
ISBN:
Category : Medicine
Languages : en
Pages : 1064
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Author: Weifei Luo
Publisher:
ISBN: 9781109838848
Category :
Languages : en
Pages : 145
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Transcription termination by RNA polymerase II (pol II) is a process that involves the release of polymerase and the RNA from the transcription elongation complex. In eukaryotes, transcription termination is tightly coupled to mRNA 3' end processing (cleavage/polyadenylation) largely via the C-terminal domain (CTD) of pol II. The torpedo model for pol II transcription termination proposes that a 5'-3' RNA exonuclease enters at the poly (A) cleavage site, degrades the 3' nascent RNA and eventually displaces polymerase from the DNA. We directly demonstrated that two exonucleases, Rat1 and Xrn1, both contribute to co-transcriptional degradation of nascent RNA but this degradation is not sufficient to cause polymerase release. Instead, Rat1 functions in both 3' end processing and termination by enhancing recruitment of 3' end processing factors. In addition, the cleavage factor Pcf11 reciprocally aids in recruitment of Rat1 to the elongation complex. How 3' end cleavage of pre-mRNA is involved in termination is still not well understood. We introduced a variant of hepatitis delta ribozyme either in the coding region or 3' flanking region of ADI-4 gene and investigated whether severing the nascent by the self-cleaving ribozyme could affect transcription termination. We showed that ribozyme cleavage within the coding region of ADH4 stimulates premature termination and dramatically enhances the recruitment of Rat1, Pcf11 and Rna15 near the ribozyme sequence although the ribozyme inserted downstream of the ADH4 poly (A) site did not affect normal termination and replacement of the ADH4 3' UTR by the ribozyme did not restore termination in the region immediately downstream. We also demonstrated that Ctk1, Ess1 and Spt5 are required for transcription termination; and Ess1 is essential for proper recruitment of Spt5 and Pcf11 of the CFIA complex at the 3' end of a gene. These observations imply that both the CTD phosphorylation and conformational modification are important to stimulate efficient termination. In summary, the results presented in this thesis suggest a unified model for pol II transcription termination: multiple contacts among the polymerase, Rat1, CFIA complex and the RNA function together to ensure efficient termination.
Author: Antoni Horst
Publisher: CRC Press
ISBN: 1351091670
Category : Science
Languages : en
Pages : 437
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Book Description
This book provides modern views of developments in medical sciences based on advances in molecular pathology. Topics discussed include the molecule; the genome of eukaryotes and its function; gene regulation; the proteins; molecular aspects of inflammation, immunology, and carcinogenesis; molecular biology of the nervous system; molecular defects in the endocrine system; molecular diseases of the blood and blood-forming tissues; and diagnosis of molecular diseases. Four tables and 75 figures illustrate the concepts and provide a quick means to reference important data. Immunologists, pathologists, geneticists, and all other researchers in the biological and medical sciences will find a wealth of information in this ground-breaking new book.
Author: Steven Lee Sanders
Publisher:
ISBN:
Category : RNA polymerases
Languages : en
Pages : 200
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Author: Joseph D. Cherayil
Publisher: CRC Press
ISBN: 9780849357442
Category : Medical
Languages : en
Pages : 182
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Book Description
This fascinating book discusses various methods that have been used from early times to the present for the isolation and characterization of total tRNA, specific tRNAs, and small molecular weight RNAs. Filled with tables and figures, it presents comparative methods and provides an overview of the progress from a historical perspective. The text features chapters on special and recent methods, isolation of tRNA from specific sources, such as chloroplasts and mitochondria, purification, and identification of modified nucleotides. It also covers suppressor tRNAs, aminoacyl tRNA synthetases, and tRNA genes. This volume is an excellent resource for all biological chemists, microbiologists, and researchers.
Author: B. D. Hames
Publisher: Oxford University Press, USA
ISBN:
Category : Music
Languages : en
Pages : 238
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Book Description
This book gives a co-ordinated review of our present knowledge of eukaryotic RNA synthesis.
