Activation of gene expression in yeast
Date
2010
Authors
Lee, Sarah Kathleen, author
Stargell, Laurie Ann, 1963-, advisor
Luger, Karolin, committee member
Nerger, Janice Lee, 1960-, committee member
Nyborg, Jennifer Kay, committee member
Paule, Marvin R., 1943-, committee member
Ross, Eric D., committee member
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Abstract
Transcription is the generation of RNA from the DNA template, and is the fundamental aspect of gene expression. As such, the initiation of transcription at genes that are transcribed by RNA polymerase II (RNAPII) is a major control point in gene expression. Organisms across the evolutionary spectrum possess genes whose transcription is regulated after recruitment of RNAPII to the promoter, or postrecruitment. This regulatory strategy has been observed in bacteria, yeast, worms, flies, and humans. Therefore, postrecruitment regulation is a conserved strategy for controlling gene expression. Genome-wide studies in Drosophila and humans demonstrate that a significant portion of these genomes are postrecruitment regulated. Recent studies in humans indicate two biologically important activators (p53 and c-myc) are involved in releasing paused polymerases from promoter DNA1,2. These regulators of cell growth and differentiation are both implicated in carcinogenesis. Thus, further understanding how activators regulate the transition from an inactive to active polymerase will prove crucial in our understanding of transcriptional regulation and human diseases. Coactivators are conserved, multiprotein complexes involved in regulating the transcription process at most genes. Yet, virtually nothing is known about the role of coactivators at postrecruitment regulated genes in yeast. The work presented in this dissertation details the identification of postrecruitment functions of two coactivators, the Mediator and SAGA complexes. My studies reveal that coactivators act as intermediaries with activator proteins to stimulate transcription after the recruitment of RNAPII to the promoter. Further, this work demonstrates that this conserved class of factors plays a role in postrecruitment regulation, a previously unappreciated aspect of coactivator function. Analysis of Mediator function at the postrecruitment regulated CYC1 gene revealed a functional submodule of the Mediator complex that is required for triggering the preloaded polymerase at the CYC1 promoter into an active polymerase. This requirement exists even when two different activator proteins control CYC1 expression, Hap2/3/4/5 and Yap1. Strikingly, this submodule is not required for activation of a recruitment regulated Yap1-dependent gene, GTT2. The Yap1 activator controls the expression of a number of genes during oxidative stress in yeast. Oxidative stress is a damaging condition that haunts all aerobic organisms, and is linked to many human ailments. Yeast respond to this biological assault with a rapid activation of many genes. My investigation of Yap1-dependent transcription demonstrated that postrecruitment regulation is more prevalent in yeast than previously thought. Analysis of SAGA function at Yap1-dependent genes revealed that Yap1 utilizes SAGA during oxidative stress. Despite a common reliance on the SAGA coactivator for expression, each gene has different specific SAGA requirements. This demonstrates an important role for the SAGA coactivator during the important biological response to oxidative stress, and the complexity inherent in transcriptional regulation. In sum, my findings illustrate the mechanisms of activated transcription yeast utilize in response to important biological stimuli. This work significantly advances our understanding of the regulation of transcription after RNAPII arrives at the promoter. It also reveals the novel role that coactivators play in stimulating transcription at the group of genes that are regulated in this fashion.
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Subject
gene transcription
RNA polymerases
transcription
Oxidative stress
SAGA
Gene expression
postrecruitment
Yeast
oxidative stress
mediator