Identification and Stoichiometric Analysis of the Monosomal Translational Complex
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University of New Hampshire University of New Hampshire Scholars' Repository Doctoral Dissertations Student Scholarship Spring 2013 Identification and stoichiometric analysis of the monosomal translational complex Xin Wang Follow this and additional works at: https://scholars.unh.edu/dissertation Recommended Citation Wang, Xin, "Identification and stoichiometric analysis of the monosomal translational complex" (2013). Doctoral Dissertations. 719. https://scholars.unh.edu/dissertation/719 This Dissertation is brought to you for free and open access by the Student Scholarship at University of New Hampshire Scholars' Repository. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of University of New Hampshire Scholars' Repository. For more information, please contact [email protected]. IDENTIFICATION AND STOICHIOMETRIC ANALYSIS OF THE MONOSOMAL TRANSLATIONAL COMPLEX BY XIN WANG B.S., Anhui Agricultural University, China 2005 DISSERTATION Submitted to the University of New Hampshire in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Genetics May 2013 UMI Number: 3572936 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Di!ss0?t&iori Piiblist’Mlg UMI 3572936 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 This dissertation has been examined and approved. C - Dissertation Directory Clyde L. Denis Professor of Biochemistry and Molecular Biology and Thomas* MyEaue Professor of Biochemistry Director of Biomolecular Interaction Technologies Center (BITC) Director of Center to Advance Molecular Interaction Science (CAMIS) PdM W. Estelle M. Hrabak Associate Professor of Plant Biology and Genetics Subhash C. Minocha Professor of Plant Biology and Genetics Jo J. Collins Associate Professor of Molecular Biology and Genetics DEDICATION I dedicate my dissertation to my family. I am especially grateful to my parents, Zhiyong Wang and Qing Liang. Their constant love, support and encouragement have sustained me throughout my life. I would also like to thank my grandparents and all other family members in China. Last but not least, I would like to say thank you and I love you to my husband, Bo Jiang. Every moment we have shared is my sweetest memory. ACKNOWLEGEMENTS I would like to express the deepest appreciation to my advisor, Dr. Clyde Denis, for his advice, encouragement and support during the past six years. Without his guidance and persistent help, this dissertation would not have been possible. Without his joking and laughing, the life in Durham, NH, would not have been as happy and wonderful. I would like to thank my committee members, Dr. Estelle Hrabak, Dr. Subhash Minocha and Dr. John Collins. Many thanks for their advice and feedback on my thesis writing. A special thank you is directed to Dr. Tom Laue, who introduced me to analytical ultracentrifugation and helped me to analyze my data. In addition, I would like to thank all the members of the Denis lab, both past and present, for their help, discussions of results and friendships. TABLE OF CONTENTS DEDICATION.......................................................................................................................iii ACKNOWLEDGMENTS.................................................................................................... iv LIST OF TABLES.................................................................................................................vii LIST OF FIGURES.............................................................................................................. viii ABSTRACT............................................................................................................................x CHAPTER PAGE GENERAL INTRODUCTION AND BACKGROUND............................................... 1 I: USE OF THE NOVEL TECHNIQUE OF ANALYTICAL ULTRACENTRIFUGATION WITH FLUORESCENCE DETECTION SYSTEM IDENTIFIES A 77S MONOSOMAL TRANSLATION COMPLEX.............................. 24 Introduction ............................................................................................................... 24 Materials and Methods ..............................................................................................26 Results........................................................................................................................31 Discussion ................................................................................................................. 41 II: STOICHOMETRIC ANALYSES OF FACTORS INVOLVED IN THE 77S MONOSOMAL TRANSLATION COMPLEX IN THE YEAST SACCHAROMYCES CEREVISIAE .........................................................................................................................44 Introduction ............................................................................................................... 44 Materials and Methods ..............................................................................................50 Results........................................................................................................................52 Discussion ................................................................................................................. 76 GENERAL CONCLUSION................................................................................................86 REFERENCES .....................................................................................................................90 APPENDIX 1.......................................................................................................................101 APPENDIX II .....................................................................................................................124 LIST OF TABLES NUMBER PAGE 1- Summary of function of PAB1 domain ........................................................................... 12 2- Absolute values for Figure 12..........................................................................................39 3- Absolute values for Figure 13 ......................................................................................... 40 4- Yeast strains used ............................................................................................................. 51 5- Relative levels of proteins in 77S complex under steady-state condition ................... 56 6- Relative levels of proteins in 77S complex at the beginning of translation initiation to that found under steady-state conditions .............................................................................. 61 7- Relative levels of proteins in 77S complex at the early stage of termination to that found under steady-state conditions ..................................................................................... 65 8- Relative levels of proteins in 77S complex following robust heat shock for 10 min to that found under non-stress conditions .................................................................................65 9- Relative levels of proteins in 77S complex under the stress of glucose depletion to that found under non-stress conditions ....................................................................................... 70 10- Abundance of 77S complex as percent of that observed in the unstressed growth condition ................................................................................................................................75 11- The absolute numbers of molecules present in the 77S complexes during the different stages of translation ............................................................................................................... 77 12- Percent of proteins in the 77S complexes during initiation and elongation conditions relative to RPS4B .................................................................................................................. 77 vii LIST OF FIGURES NUMBER PAGE 1- General Eukaryotic Cap-Dependant Translation Initiation Model ...................................4 2- The life cycle of cytoplasmic mRNA ................................................................................ 9 3- Structure of poly(A) binding protein ................................................................................12 4- The detailed primary structure of eIF4G l ....................................................................... 14 5- Western assay of Flag-PABl pulldown of strains containing PAB1 variants .............. 18 6- Commonly used two-channel cell in absorbance optical system ...................................21 7- Parameters setting in SEDFIT software for AU-FDS analysis .....................................29 8- Effect of sodium azide on 77S complex abundance ....................................................... 32 9- Effect of oxidative stress on 77S complex abundance ................................................... 33 10- Cycloheximide blocks ribosome runoff under glucose depletion ................................35 11- Effects of