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Changes in the Rpb3 Interactome Caused by the Deletion of Rpb9

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Changes in the Rpb3 Interactome Caused by the Deletion of Rpb9 CHANGES IN THE RPB3 INTERACTOME CAUSED BY THE DELETION OF RPB9 IN SACCHAROMYCES CEREVISIAE Eric A Talbert Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree Master of Science in Biochemistry and Molecular Biology Indiana University August 2016 Accepted by the Graduate Faculty, Indiana University, in partial fulfillment of the requirements for the degree of Master of Science Master’s Thesis Committee ________________________________________ Amber L. Mosley, PhD. Chair ________________________________________ Mark G. Goebl, PhD. ________________________________________ Andy Hudmon, PhD ii ACKNOWLEDGEMENTS I wish to extend my heartfelt thanks to the following people for their aid completing this project. It would not have been possible without their help. To Dr. Amber Mosley- For taking me in and helping me make the best of a bad situation, and for being patient and kind when that was the most important thing in the world. To Dr. Mark Goebl- For being a constant source of guidance and mentorship throughout my time with the Biochemistry Department. To Dr. Andy Hudmon- For being a teacher and mentor, and also a friend. To my colleagues in the Mosley Lab, Whitney Smith-Kinnaman, Dr. Melanie Fox, Dr. Jerry Hunter, Sarah Peck, and Jose Victorino- For your help and instruction, as well as the use of your data. To my parents, Larry and Margaret Talbert- For your unwavering love and support throughout my life. To my sister, Dr. Erin Talbert- For your advice and mentorship, and for lending an ear when I needed one. iii Eric A Talbert CHANGES IN THE RPB3 INTERACTOME CAUSED BY THE DELETION OF RPB9 IN SACCHAROMYCES CEREVISIAE RNA Polymerase II (Pol II) is the primary actor in the transcription of mRNA from genes. Pol II is a complex composed of twelve protein subunits. This study focused on the changes in the interactome of Rbp3 in S. cerevisiae when the Pol II subunit Rpb9 is removed. Rpb3 is one of the core subunits of Pol II, and any significant changes to the Rpb3 incteractome due to the loss of Rpb9 can be used to infer new information about Rpb9’s role in the Pol II complex. Rpb3 was pulled down using FLAG purification from both wild type and rpb9Δ S. cerevisiae cultures. Rpb3 and the proteins complexed with it were then analyzed using multi-dimensional protein identification technology (MudPIT), a form of liquid chromatography-mass spectrometry (LC-MS). This data was searched using the SEQUEST database search algorithm, and the results were further analyzed for likelihood of interaction using Significance Analysis of INTeractome (SAINT), as well as for post-translational phosphorylation. Deletion of rpb9 did not present any changes in Pol II phosphorylation however it did cause several changes in the interaction network. The rpb9Δ strain showed new interactions with Rtr1, Sen1, Vtc4, Pyc1, Tgl4, Sec61, Tfb2, Hfd1, Erv25, Rib4, Sla1, Ubp15, Bbc1, and Hxk1. The most prominent of these hits are Rtr1, an Rpb1 C-terminal domain phosphatase linked to transcription termination, and Sen1, an RNA/DNA nuclease that terminates transcription. In addition, this mutant showed no interaction with Mtd1, an interaction that is present in the wild type. In all cases, these hits should be considered fuel for future research, rather than conclusive evidence of novel interactions. Amber L Mosley, PhD Chair iv TABLE OF CONTENTS LIST OF TABLES ............................................................................................................................... vi LIST OF FIGURES ............................................................................................................................. vii INTRODUCTION Genes and Gene Expression............................................................................................................. 1 Transcription .................................................................................................................................... 3 RNA Polymerase II Function ............................................................................................................. 9 MATERIALS AND METHODS Bioinformatics ................................................................................................................................ 13 Affinity Purification ........................................................................................................................ 13 Multidimensional Protein Identification Technology .................................................................... 14 Peptide Spectra Analysis ................................................................................................................ 16 Significance Analysis of INTeractome ............................................................................................ 16 Isolation of Protein Fraction from FLAG Tagged S. cerevisiae ....................................................... 17 Pull-Down and Digestion of Protein Fraction ................................................................................ 18 MudPIT Analysis ............................................................................................................................. 18 Analysis of MS/MS Spectra ............................................................................................................ 19 RESULTS Phosphorylation Analysis ............................................................................................................... 21 SAINT Analysis of Pol II ................................................................................................................... 22 Significant Hits from SAINT Analysis .............................................................................................. 25 DISCUSSION ................................................................................................................................... 30 CONCLUSION ................................................................................................................................. 40 APPENDIX 1: SAINT TABLE ............................................................................................................ 41 REFERENCES ................................................................................................................................... 74 CURRICULUM VITAE v LIST OF TABLES 1. Selected Actors in mRNA Transcription .................................................................................... 12 2. Reagents and Media ................................................................................................................. 18 vi LIST OF FIGURES 1. Transcription of Messenger RNA ................................................................................................ 4 2. Initiation of Messenger RNA Transcription ................................................................................. 6 3. Changes in Phosphorylation State of Serine-5 of the RNA Polymerase II CTD Heptapeptide .... 8 4. Schematic of RNA Polymerase II Holoenzyme .......................................................................... 11 5. Schematics of Wild Type and rpb9Δ Pol II ................................................................................ 24 6. SP Values of WT and rpb9Δ strains for Pol II Subunits and Significant Hits .............................. 24 7. Changes in the Protein Network of Rpb3 between Wild Type and rp9Δ Cells ......................... 31 vii INTRODUCTION Genes and Gene Expression Transcription is the process of transforming genetic information stored within DNA into functional RNA molecules. These molecules perform a variety of roles within the cell, from structural and catalytic functions in ribosomes to blueprints for the synthesis of proteins. In all cases, however, transcription starts with a DNA template. DNA is a polymer of nucleotides that serves as the primary information storage medium for living cells. DNA chains form a distinctive double-helix shape, with two phospho-sugar chains linked together by nitrogenous bases, forming a set of rungs on the twisted ladder of the DNA molecule. The sequence of these base pairs makes up a code that contains the actual genetic information stored within DNA (Watson, 1953). A section of DNA that codes for a specific functional product is called a gene. Genes are the primary target of transcription. Genes can be divided into several sections based on function. Most notable of these is the transcribed region, which is the stretch of DNA that is copied during transcription. The first point of the transcribed region is called the transcription start site (TSS). Promoters are regions upstream of the TSS that interact directly or indirectly with the transcription complex, the group of proteins that, when bound together, perform transcription (Bayev, 1980). Most genes contain a core promoter region just upstream of the TSS. This is the binding site of the preinitiation complex, a grouping of proteins that work together to initiate transcription. Along with this core promoter, many promoter regions also contain enhancer elements that are bound by transcriptional activators, which are other proteins that encourage transcription of the gene (Bran, 1985). 1 Much of an organism’s genome functions to regulate gene expression, or how and when genes are transcribed. In addition to transcriptional activators discussed above, some of this control comes from how DNA molecules are organized into structures called chromosomes.
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