Author: Rachel Naomi Fish
Publisher:
ISBN:
Category :
Languages : en
Pages : 622

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Author: Karen Renee Christie
Publisher:
ISBN:
Category :
Languages : en
Pages : 600

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Regulation of the process of transcriptional elongation is an important control mechanism in the expression of some genes. To fully understand this form of regulation will require better understanding of the functions of transcription elongation factors. The goal of this work was to characterize the transcription elongation factor TFIIS from Saccharomyces cerevisiae, originally called P37. I demonstrated that, like the mammalian TFIIS proteins, the yeast protein stimulates RNA polymerase II to cleave the nascent RNA transcript and to read-through an intrinsic block to elongation. Investigation of the protein-protein contacts between TFIIS and RNA polymerase II indicated that the carboxyl-terminal domain of the largest subunit, subunit four, and subunit seven of the polymerase are not required for TFIIS to promote cleavage and read-through by the polymerase. In addition the carboxyl-terminal half of the yeast TFIIS protein is sufficient for both of these in vitro activities. This result is consistent with the previous results demonstrating the carboxyl-terminus of mouse TFIIS was sufficient to activate RNA polymerase in vitro.

Author: Vanessa Cheung
Publisher:
ISBN:
Category :
Languages : en
Pages :

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Previous studies in Saccharomyces cerevisiae have shown that mutations in SPT6, SPT16, SET2, and other genes encoding particular chromatin and transcription factors cause the activation of cryptic promoters within coding regions, resulting in the expression of short RNA transcripts. In this work, we examine cryptic initiation in S. cerevisiae on a genome-wide scale to identify all factors required for repressing cryptic promoters and to determine the extent and possible role of cryptic initiation. Using large-scale mutant selections and synthetic genetic array analysis, we have identified mutants affecting histone gene regulators, chromatin remodeling factors, and histories as allowing cryptic initiation. Through microarray analysis, we show that approximately 1,000 genes, or almost one-sixth of the S. cerevisiae genome, express short transcripts in spt6 or spt16 mutants. A significant number of these genes are shown to be conserved with respect to synonymous substitution rates in the transcribed regions corresponding to the short transcripts. Furthermore, we show that short transcripts are capable of being translated into corresponding short proteins. Finally, we show that cryptic initiation can occur in wild-type cells following a nutritional shift from rich to minimal media. Our results demonstrate that maintenance of chromatin structure and modifications is important for repressing a large number of cryptic promoters distributed throughout the genome and suggest that cryptic initiation has the potential to play a physiological role in expressing alternate genetic information in response to environmental changes.

Author: Chen Yang
Publisher:
ISBN:
Category :
Languages : en
Pages : 213

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The basal eukaryotic transcription machinery for protein coding genes is highly conserved from yeast to high eukaryotes. However, while human cells usually initiate at a single transcription start site approximately 30 bp downstream of a TATA element, Schizosaccharomyces pombe typically initiates at multiple sites 30-70 bp, and Saccharomyces cerevisiae 40 to 200 bp downstream of the TATA. The determinant factor(s) for the species specific initiation and the underlying mechanisms for the multiple far downstream start site utilization in yeast are not well understood. By swapping the highly purified transcription factors between S. pombe and S. cerevisiae reconstituted transcription systems, we confirmed previous observations that RNA polymerase II and/or the general transcription factor TFIIB determine the species-specific start site utilization patterns. Further genetic and biochemical assays of TFIIB chimeras indicated that RNAPII, but not TFIIB as previously proposed, determines the distinct initiation patterns not only between the two yeast systems but also between human and yeast systems. Bubble template initiation assays showed that there is an inverse correlation between the amount of negative charge in the TFIIB B-fingertip and the efficiency of the first phosphodiester bond formation. Moreover, biochemical studies indicate that multiple initiation steps, including first phosphodiester bond formation, and RNA:DNA hybrid stability determined initiation-to-elongation transition, could be modulated to regulate the far downstream start sites utilization in S. cerevisiae. A model for multiple far downstream transcription start sites formation in S. cerevisiae is proposed.

Author:
Publisher:
ISBN:
Category :
Languages : en
Pages : 204

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Transcription of protein-coding genes by eukaryotic RNA polymerase II (RNAPII) is a multistep process that involves the concerted action of RNAPII and accessory proteins including the general transcription factors (GTFs); TFIID, TFIIB, TFIIF, TFIIE and TFIIH. The GTFs are being intensely studied to determine their function during the different stages of the transcription cycle. Numerous investigations have reported functions for the general transcription factor IIF (TFIIF) of higher eukaryotes in multiple stages of the transcription cycle, although few studies have examined the TFIIF homolog in the yeast Saccharomyces cerevisiae. The objective of this dissertation is to better understand the mechanism of action of S. cerevisiae TFIIF during the different stages of the RNAPII transcription cycle. Although the basal transcription factors are highly homologous in eukaryotes, the mechanism of transcription start site utilization on TATA-dependent promoters in S. cerevisiae is fundamentally different from that in higher eukaryotes, where the PIC assembles on the promoter and transcription initiates at a discrete site 25-30 base pairs downstream of the TATA element with the architecture of the PIC determining the initiation site. In contrast, the S. cerevisiae RNAPII machinery typically initiates at multiple sites in a window from 45 to 120 base pairs downstream of the TATA element. Results in this thesis support a transcription initiation mechanism that involves transcription-independent translocation of the yeast RNAPII to the far downstream start sites. Previous work in our laboratory identified mutations in the yeast TFIIF subunits Tfg1 and Tfg2 that confer upstream shifts in start site utilization. In vivo and in vitro studies demonstrate that TFIIF modulates the utilization of transcription start site sequences through its interaction with RNAPII. In the second and third part of this dissertation, genetic and biochemical approaches were utilized to better define TFIIF functions at post-initiation steps in the S. cerevisiae transcription cycle, and support a role for TFIIF in both promoter escape and early elongation. Combined results of this work support a model for TFIIF function where the TFIIF, through its interaction with RNAPII, affects the DNA recognition properties of the polymerase during start site utilization, promoter escape, and early elongation.

Author: Jeffrey N. Strathern
Publisher:
ISBN:
Category : Science
Languages : en
Pages : 776

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Author: Caren Jody Stark
Publisher:
ISBN:
Category :
Languages : en
Pages : 538

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Author: Ron Milo
Publisher: Garland Science
ISBN: 1317230698
Category : Science
Languages : en
Pages : 400

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Book Description
A Top 25 CHOICE 2016 Title, and recipient of the CHOICE Outstanding Academic Title (OAT) Award. How much energy is released in ATP hydrolysis? How many mRNAs are in a cell? How genetically similar are two random people? What is faster, transcription or translation?Cell Biology by the Numbers explores these questions and dozens of others provid

Author: David C. Amberg
Publisher: CSHL Press
ISBN: 0879697288
Category : Genetics
Languages : en
Pages : 250

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Book Description
"Methods in Yeast Genetics" is a course that has been offered annually at Cold Spring Harbor for the last 30 years. This provides a set of teaching experiments along with the protocols and recipes for the standard techniques and reagents used in the study of yeast biology.

Author: Jeffrey S. Smith
Publisher: Humana
ISBN: 9781493951826
Category : Science
Languages : en
Pages : 0

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Book Description
Yeast Genetics: Methods and Protocols is a collection of methods to best study and manipulate Saccharomyces cerevisiae, a truly genetic powerhouse. The simple nature of a single cell eukaryotic organism, the relative ease of manipulating its genome and the ability to interchangeably exist in both haploid and diploid states have always made it an attractive model organism. Genes can be deleted, mutated, engineered and tagged at will. Saccharomyces cerevisiae has played a major role in the elucidation of multiple conserved cellular processes including MAP kinase signaling, splicing, transcription and many others. Written in the successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, Yeast Genetics: Methods and Protocols will provide a balanced blend of classic and more modern genetic methods relevant to a wide range of research areas and should be widely used as a reference in yeast labs.