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Open Jordanirvinthesis2.Pdf The Pennsylvania State University The Graduate School Department of Biochemistry, Microbiology, and Molecular Biology GENOME-WIDE FUNCTIONAL ANALYSIS OF TAF1 AND MOT1, REGULATORS OF THE TATA BOX BINDING PROTEIN IN SACCHAROMYCES CEREVISIAE A Thesis in Biochemistry, Microbiology and Molecular Biology by Jordan Davis Welch Irvin © 2005 Jordan Davis Welch Irvin Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2005 The thesis of Jordan Davis Welch Irvin was reviewed and approved* by the following: B. Franklin Pugh Professor of Biochemistry and Molecular Biology Thesis Advisor Chair of Committee Song Tan Associate Professor of Biochemistry and Molecular Biology Richard J. Frisque Professor of Molecular Virology Blake R. Peterson Associate Professor of Chemistry Jerry L. Workman Investigator, The Stowers Institute for Medical Research Kansas City, MO Special Member Robert A. Schlegel Professor of Biochemistry and Molecular Biology Head of the Department of Biochemistry, Microbiology and Molecular Biology *Signatures are on file in the Graduate School iii ABSTRACT The TATA box binding protein (TBP) is a key protein in the process of gene expression, or transcription. The binding of TBP to DNA nucleates the formation of the pre-initiation complex of transcription factors at a gene. Numerous transcription factors regulate TBP both positively and negatively. TBP directs transcription from two multi- subunit complexes, SAGA and TFIID, that operate on separate classes of genes. The studies described here dissect the contributions that distinct domains of two transcription factors, TAF1 and MOT1, make on a genome-wide scale. TAF1 is an essential subunit of TFIID with several known activities, including binding TBP. MOT1 has been linked to the SAGA co-activator complex through genetic, biochemical, and genome-wide studies, and is known to remove TBP from DNA via ATP hydrolysis. We find that all activities of TAF1 tested are required for proper expression of TFIID-dominated genes (~90% of the genome), and a domain of TAF1 plays a role in preventing TAF1 from associating with SAGA dominated genes. Deletion of functional domains of TAF1 contribute to protein instability, dominant toxicity, and can not support viability as the sole source of TAF1. In contrast to the results from TAF1, several distinct patterns of MOT1-mediated regulation emerge. MOT1 appears to be a major regulator of transcription at a small subset of yeast genes. A genome-wide assessment indicates MOT1 acts primarily to activate TFIID-dominated (housekeeping) genes and repress SAGA-dominated (stress response) genes. The N-terminus of MOT1, which interacts with TBP is necessary for repression of stress response genes. A subset of genes depends on most or all of the iv regions of MOT1 that were mutated for proper expression. Interestingly, the TBP- associated complex NC2, which makes positive and negative contributions to gene expression, appears to work in concert with MOT1 to regulate TBP. At genes where MOT1 is acting positively, NC2 also plays an activating role. Where MOT1 exerts repressive effects, NC2 also works to down-regulate expression. v TABLE OF CONTENTS LIST OF FIGURES .....................................................................................................vii ABBREVIATIONS .....................................................................................................ix ACKNOWLEDGEMENTS.........................................................................................xi Chapter 1 Introduction ................................................................................................1 1.1 Overview of Gene Regulation ........................................................................1 1.2 Regulation of RNA Polymerase II Expression...............................................3 1.3 Chromatin Structure and Gene Expression.....................................................5 1.4 Chromatin Remodeling Complexes................................................................6 1.5 Histone Acetylation ........................................................................................8 1.6 The SAGA Co-Activator Complex ................................................................8 1.7 General Transcription Factors ........................................................................10 1.7.1 TFIID....................................................................................................10 1.7.1.1 TFIID Subunit TAF1..................................................................13 1.8 Bromodomain Factor 1...................................................................................15 1.9 The TATA Box Binding Protein (TBP) .........................................................16 1.10 MOT1 ...........................................................................................................16 1.11 The Yeast NC2 Complex..............................................................................18 1.12 Bibliography .................................................................................................20 Chapter 2 Interaction of the Amino-Terminal Domain of yTAF1 and TBP ..............39 2.1 Introduction.....................................................................................................39 2.2 Identification of TAF1 N-Terminal Domain (TAND) and Association with TBP........................................................................................................40 2.3 TAND Homology ...........................................................................................42 2.4 Structure of TBP.............................................................................................43 2.5 TAND Structure Mimics the TATA Box .......................................................48 2.6 Mutagenesis of TBP and effects on TAND binding.......................................51 2.7 Results.............................................................................................................53 2.8 Discussion.......................................................................................................60 2.9 Materials and Methods ...................................................................................64 2.9.1 Subcloning of F23K and F23K/D66K Mutations........................................64 2.9.1 GST-TAND purification..............................................................................65 2.9.1 Purification of 6His-TBP mutants ...............................................................66 2.9.1 GST Pulldowns............................................................................................67 2.9.1 Molecular Structrues....................................................................................68 2.10 Bibliography .................................................................................................70 Chapter 3 Genome-Wide Roles of TAF1 Functional Domains on Transcription ......78 vi 3.1 Abstract...........................................................................................................78 3.2 Introduction.....................................................................................................79 3.3 Results and Discussion ...................................................................................82 3.3.1 Mutant Design .............................................................................................82 3.3.1 Growth Phenotypes......................................................................................89 3.3.1 Subcellular Localization ..............................................................................91 3.3.1 Protein Stability ...........................................................................................94 3.3.1 Genome-wide Expression............................................................................96 3.3.1 Future Directions .........................................................................................104 3.4 Materials and Methods ...................................................................................105 3.4.1 Plasmids.......................................................................................................105 3.4.1 FHT-TAF1 mutant strains ...........................................................................106 3.4.1.1 Haploids .....................................................................................107 3.4.1 PCR..............................................................................................................108 3.4.1 Cell Growth/Lethality/Dominant Toxicity Assays......................................111 3.4.1 Galactose Shutoff.........................................................................................111 3.4.1 Immunofluorsecence....................................................................................112 3.4.1 Western Blotting..........................................................................................113 3.4.1 Galactose Additiong Timing........................................................................113 3.4.1 Microarray Analysis ....................................................................................114 3.5 Bibliography ...................................................................................................116 Chapter 4 Genome-wide Analysis of Functional Domains of Yeast Transcription Factor MOT1 ........................................................................................................121
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