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ROLE of RIBOSOMAL PROTEIN Us9/Ys16 in TRANSLATION INITIATION AND

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ROLE of RIBOSOMAL PROTEIN Us9/Ys16 in TRANSLATION INITIATION AND ROLE OF RIBOSOMAL PROTEIN uS9/yS16 IN TRANSLATION INITIATION AND ELONGATION IN YEAST Saccharomyces cerevisiae. SUPRIYA JINDAL Bachelor of Science in Biochemistry Aligarh Muslim University July 2008 Master of Science in Biochemistry Aligarh Muslim University July 2010 Submitted in partial fulfillment of requirement for the degree DOCTOR OF PHILOSOPHY IN REGULATORY BIOLOGY with specialization in Cellular and Molecular Medicine at the CLEVELAND STATE UNIVERSITY May 2019 @SupriyaCopywright We hereby approve this dissertation for Supriya Jindal Candidate for the Doctor of Philosophy in Regulatory Biology degree for the Department of Biological, Geological and Environmental Sciences and the CLEVELAND STATE UNIVERSITY College of Graduate Studies Dissertation Chairperson, Dr. Anton A. Komar BGES, 02/11/2019 Dissertation Committee Member, Dr. Barsanjit Mazumder BGES, 02/11/2019 Dissertation Committee Member, Dr. G. Valentin Börner BGES, 02/26/2019 Dissertation Committee Member, Dr. Donna Driscoll Cleveland Clinic, 01/22/2019 Dissertation Committee Member, Dr. Girish C. Shukla BGES, 02/23/2019 Dissertation Committee Member, Dr. William Merrick Case Western Reserve, 01/23/2019 Student’s Date of Defense: November 29, 2018 Dedicated to my parents and my brother ACKNOWLEDGEMENTS First and foremost I would like to acknowledge and sincerely thank Dr. Anton A. Komar for being my PhD mentor. His continued guidance, support and encouragement made it possible to successfully finish this work. He would not just give direction to my research but constantly motivated me to think and try things on my own. He always supported me through my thick and thin, whether it was losing my father or having my daughter, and yet did not let me lose sight of my research goals. His commitment, diligence and dedication towards his work will always be an inspiration for me. I believe under his mentorship, I learnt not only the necessary research skills but also the confidence to be able to solve a problem, be whatever area of life it is. I thank Dr. G. Valentine Bӧrner, Dr. Barsanjit Mazumder and Dr. Donna Driscoll for being a part of my advisory committee and giving their valuable time, suggestions and criticisms, which significantly improved my thesis work. Dr. Girish C. Shukla and Dr. Wiliam Merrick for agreeing to be a part of my dissertation defense committee and all the faculty member of the department and graduate program for their help as needed. I would also like to express my thankfulness to the graduate program for giving me the opportunity to teach as teaching assistant which was a very valuable experience. I would like to acknowledge the Cellular and Molecular Medicine Specialization program for two years funding support towards this work and Cleveland State University for providing the Doctoral Dissertation Research Expense Award Fellowship to finish my doctoral studies. I would like to acknowledge and thank my lab mates Sujata Jha, Arnab Ghosh and Nishant Singh for the initial guidance and training that I received in the lab. Also my thanks to the help and support I received from other members of the lab Puja Nanavaty, Artyem and Aanchal Agrawal over the years. A special thanks to Amra Ismail, who helped me in the last phase of my experiments while I was pregnant and delivered my baby. I would like to thank all the students, post docs and research assistants (both past and present) from the department for their help during the years with special mention to Sonal, Subhra, Kuldeep, Nikhil, Rima, Jey, Savita, Jayeeta, Jagjit, and Amit. I really valued our scientific discussions and experience sharing on many occasions, as well as the help I received in letting me borrow things many times from your lab for my thesis work. I would also like to thank the TA‟s of the department for their help on many occasions especially Subhra, Aanchal, Amra, and Ali. I would like to thank Dr. Girish Shukla for many friendly yet insightful discussions, and guidance, and going out of his way to help me in many occasions; Dr. Crystal M. Weyman for numerous help; Dr. Donald Lindmark and Dr. Ralph Gibson for their help when I was working as a teaching assistant under their supervision. I would also like to thank all the staff members of Department of Biological, Geological and Environmental Sciences and Cleveland State University (especially Michelle Zinner, Suzie Moreno, Monica Warner and Carolee Pichler) for their help and support. I thank Cleveland State University, College of Sciences and Health Professions, Department of Biological, Geological and Environmental Sciences, Cellular and Molecular Medicine Program and Center for Gene Regulation in Health and Disease for numerous funding and facilities, which were essential for the success of my work. Finally, my acknowledgements to my family: my (late) father and my mother who were my first teachers in life and who laid a strong foundation for my upbringing and education, my brother who always supported and encouraged me to be successful and independent, my husband who always stood strong with me and supported me in every possible way and, my 5 months old daughter who has been very cooperative and patient with me. Last, but not the least, my spiritual teacher Sri Sri: his knowledge and wisdom has been my life jacket in the toughest of the moments, who shows me things and life in bigger perspective and continuously inspires me to work selflessly and contribute to the world. ROLE OF RIBOSOMAL PROTEIN uS9/yS16 IN TRANSLATION INITIATION AND ELONGATION IN YEAST Saccharomyces cerevisiae. SUPRIYA JINDAL ABSTRACT The process of translation in all living cells is performed by ribosomes and is divided into four major steps (initiation, elongation, termination and ribosome recycling). Ribosomes consist of ribosomal RNA (rRNA) and proteins and are composed of two subunits small and large. There are three major sites for tRNA binding within ribosome: the (aminoacyl) A-site which accepts the aminoacyl (aa)-tRNA; the P-site, where the peptidyl-tRNA is formed and the (exit) E-site, where deacylated tRNA exits the ribosome. These sites are formed by both rRNA and ribosomal proteins. Though rRNA are involved in the catalysis of protein synthesis, the contribution of individual ribosomal proteins to protein synthesis is not fully understood in molecular terms. Yeast ribosomal protein uS9/yRps16, is universally conserved and is located on the solvent side of the small ribosomal subunit. It has a long protruding C-terminal tail (CTT) which extends towards the mRNA cleft. This CTT contributes to the formation of the ribosomal P-site. uS9/yRps16 last positively charged C terminal residue (Arg), is invariably conserved and is believed to enhance interaction of the negatively charged initiator tRNA, when base-paired to AUG codon in the P site. However, biochemical evidence in support of this notion is limited. Our biochemical analysis of the uS9 mutants showed that the C terminus of uS9 plays an vii important role during translation initiation. We found that uS9 C-terminal residues (their exact location and nature) are critical for efficient recruitment of the eIF2•GTP•Met- Met tRNAi ternary complex and for responding properly to an AUG codon in the P-site, during scanning phase of initiation. These residues also regulate hydrolysis of GTP (from Met eIF2•GTP•Met-tRNAi complex) to GDP and Pi. Furthermore, deletion of the last two residues of uS9 CTT, exhibits resistance to anisomycin, decreased association of elongation factor eEF1A to polyribosomes at the A-site and decreased programmed ribosomal frameshift (PRF) efficiency, thus showing that uS9 C terminal region modulates elongation fidelity. Therefore, we propose that uS9 CTT is critical for proper control of the complex interplay of events surrounding accommodation of initiator and elongator tRNAs in the P- and A-sites of the ribosome. viii TABLE OF CONTENTS Page ABSTRACT ...................................................................................................................... vii LIST OF TABLES .......................................................................................................... xiii LIST OF FIGURES ......................................................................................................... xiv LIST OF ABBREVIATIONS ........................................................................................ xiiv CHAPTER I. INTRODUCTION ............................................................................................... 1 1.1 Overview of translation ................................................................. 1 1.2 Translation machinery .................................................................... 2 1.2.1 Ribosomes (Composition and Evolution) .......................... 2 1.2.2 Translation factors and their evolution ............................... 8 1.3 Eukaryotic translation initiation .................................................. 15 1.4 Translation reinitiation ................................................................. 23 1.5 Eukaryotic translation elongation ................................................. 27 1.6 Programmed ribosomal frameshifting .......................................... 31 1.6.1 Programmed -1 ribosomal frameshifting .......................... 31 1.6.2 Programmed +1 ribosomal frameshifting .......................... 32 1.7 Ribosomal proteins ....................................................................... 34 1.7.1 Importance of ribosomal proteins ....................................
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