Characterization of Ribosomes and Ribosome Assembly Complexes by Mass Spectrometry A dissertation submitted to the Graduate School of the University of Cincinnati In partial fulfillment of the requirements for the degree of Doctor of Philosophy (Ph.D.) In the Department of Chemistry of the College of Arts and Sciences 2013 By Romel P. Dator M.S. Chemistry, University of the Philippines Diliman, 2008 B.S. Chemistry, University of the Philippines Visayas, 2003 Committee Chair: Prof. Patrick A. Limbach Abstract The biogenesis and assembly of the ribosome involves a coordinated cascade of events including rRNA processing, folding, and post-transcriptional modifications of rRNA along with the association of ribosomal proteins (r-proteins). Unraveling the pathways and dynamics of these complex structural processes is a significant challenge. While conventional biophysical techniques such as nuclear magnetic resonance (NMR), X-ray crystallography and cryo-electron microscopy (cryo-EM) provide high-resolution information, these methods are not ideally suited to characterize transient and heterogeneous ribosome assembly intermediate structures. Here mass spectrometry-based approaches are being used to gain insights into the composition and structural organization of ribosomes and ribosome assembly particles in vivo, particularly those particles that result from perturbations (e.g. deletion of assembly factors, antibiotics). 15N-labeling and data-dependent LC-MS/MS were used to characterize the proteins associated with pre-30S complexes from E. coli RimM and RbfA deletion strains. RimM and RbfA are ribosome assembly factors implicated in the maturation of the small 30S subunit in bacteria. The precise roles of these assembly factors in 30S subunit assembly are unclear. Along with in vivo x- ray footprinting and mass spectrometry data, detailed molecular mechanisms how RimM and RbfA facilitate maturation of the 30S subunit in vivo were uncovered. Although relative quantitation of proteins by 15N-labeling and LC-MS/MS provides information on the differential expression of proteins in normal and perturbed samples, this approach is limited to comparing two samples at a time, labeling can be expensive and laborious, and not amenable to other multicellular organisms. The applicability of a label-free approach, LC-MSE for absolute “ribosome-centric” quantification of r-proteins was evaluated. Using an additional dimension of gas-phase separation through ion mobility and multiple endoproteinase digestion allow accurate and reproducible quantitation of proteins associated with mature ribosomes. The improved LC- i MSE approach was then extended to characterizing proteins associated with different functional states of the ribosomes (free 30S, free 50S, 70S and polysomes). The actively translating ribosomes (polysomes) contain stoichiometric amounts of proteins consistent with their known stoichiometry within the complex. Significant heterogeneity was found with free subunits as they are composed of immature complexes and dissociated subunits from 70S. The stoichiometric measurements among the different classes of ribosomes showed very good run-to-run reproducibility and biological reproducibility with %CV less than 15% and 35%, respectively. Finally, the in vivo assembly complexes formed in the presence of the antibiotic erythromycin was isolated and characterized. A strategy was devised to isolate and purify the erythromycin-induced 50S assembly particle in SK5665 cells grown in the presence of the antibiotic erythromycin. Quantitative analysis of the proteins associated with the Δ50S particles suggests a heterogeneous collection of 50S intermediates with different subsets and varying amounts of proteins. The amounts of the assembly factors, SrmB and DbpA, detected in the Δ50S particle indicate that the Δ50S particle is immature and is a late assembly intermediate. ii iii Acknowledgments I would like to express my sincerest gratitude to my advisor, Professor Patrick A. Limbach, for giving me this great opportunity to work under his guidance, supervision, and for the countless opportunities to grow and develop as a scientist. Thank you for the enlightening intellectual discussions and for inspiring me to pursue bigger scientific possibilities. I would like to thank my dissertation committee members, Professor Albert Bobst and Professor Joseph Caruso, for their guidance, constructive criticisms to improve my scholarly work, and for being part of my professional development. A big thank you to my collaborators, Professor Sarah Woodson and Sarah Clatterbuck Soper (Johns Hopkins University) for a very successful collaborative work. To my colleagues and mentors, Dr. Kirk Gaston, Dr. Balu Addepali, and Dr. Anne McLachlan for sharing me their expertise, help for the techniques I am not so familiar with, and most importantly for the fruitful scientific discussions. The current and past Limbach Group members for making my research life fun, enjoyable, and highly stimulating. I would like to thank Professor Ken Greis, Dr. Wendy Haffey, and Therese Rider (UC Department of Cancer Biology) for allowing me to use their MALDI MS instrument, access to the Mascot Server for my proteomics analysis, and for the technical assistance. To Dr. Stephen Macha and Dr. Larry Sallans (Mass Spectrometry Facility) for their help and guidance when I need to run my samples in the facility’s mass spectrometers. My heartfelt thanks to Dr. Ann P. Villalobos for being instrumental to my graduate school career and for the enormous help, support and encouragement all these years. To my master’s advisors, Dr. Sonia Jacinto and Dr. Amelia Guevara for motivating me to pursue graduate studies in US and for paving my intellectual curiosity to advance my career in science. To my best friend, soon to be Dr. Morwena Jane V. Solivio, for being a fun and enjoyable buddy and for the great memories we have had all these years. To my family and friends, for the love and support and for inspiring me to become a better and compassionate person. I am thankful for the National Institutes of Health (NIH) for the funding and the Chemistry Graduate Student Association (CGSA) for giving me the opportunity to serve and to explore my leadership capabilities. My thanks and appreciation to the UC Department of Chemistry, faculty and staff and the University of Cincinnati for giving me this once in a lifetime opportunity to pursue graduate studies here and for being part of my scientific aspirations. iv Table of Contents Abstract…………………………………………………………………………….................................i Acknowledgments………………………………………………………………….………….............iv List of Tables………..………………………………………………………………….......................vii List of Figures……………………………….……………………………………................................ix List of Schemes………………………………….……………………………………………………xiv List of Abbreviations…………………………….…………………………………………................xv Chapter 1. Introduction and Background…………………………………………………………....1 1.1 Ribosome Structure, Function, and Antibiotic Action……………………….……….…....1 1.1.1 Ribosome Structure………………………………………………………….......1 1.1.2 Ribosome Function……………………………………………………………...4 1.1.3 Ribosome as Target of Antibiotics…………………………………….………...5 1.2 Bacterial Ribosome Biogenesis and Assembly……………………………………….........6 1.2.1 Posttranscriptional Modifications of Ribosomal RNA………………………….7 1.2.2 Posttranslational Modification of Ribosomal Protein…………………….……..9 1.2.3 In Vitro and In Vivo Studies of Ribosome Assembly……………………….....10 1.2.4 Ribosome Assembly Factors……………………………………………….......14 1.2.5 Ribosome Assembly as Target of Antibiotics…………………………...……..17 1.3 Biophysical and Biochemical Methods to Study Ribosome Assembly………..................19 Chapter 2. Literature Review…..........................................................................................................22 2.1 Characterization of Ribosomal Proteins by Mass Spectrometry……………………….....22 2.1.1 Identification of Ribosomal Proteins Their Posttranslational Modifications…..25 2.1.2 Analysis of Ribosomal Proteins to Probe Ribosome Topology………………..28 2.2 Mass Spectrometry-Based Quantitative Analysis of Ribosomal Proteins………………...29 2.2.1 Stable Isotope Labeling for Relative and Absolute Quantitation…………........31 2.2.2 Label-Free Approaches for Relative and Absolute Quantitation…………........32 v 2.3 Purpose of the Work Presented……………………………………………………............35 Chapter 3. Analysis of 30S Ribosomal Subunit Assembly Particles in RimM and RbfA Escherichia coli Deletion Mutants by Mass Spectrometry 3.1 Introduction……………………………………………………………..............................38 3.2 Experimental………………………………………………………………………............38 3.3 Results and Discussion…………………………………………………………….............43 3.4 Conclusion………………………………………………………………………...............53 Chapter 4. Quantitation of Bacterial Ribosomal Proteins by LC-MSE 4.1 Introduction……………………………………………………………………………......54 4.2 Experimental………………………………………………………………………............57 4.3 Results and Discussion………………………………………………………………….....61 4.4 Conclusion………………………………………………………………….......................71 Chapter 5. Characterization of Ribosomal Proteins Associated with the Different Functional States of the Ribosome 5.1 Introduction……………………………………………………………………………......73 5.2 Experimental…………………………………………………………………………........74 5.3 Results and Discussion……………………………………………………………….........75 5.4 Conclusion………………………………………………………………….......................87 Chapter 6. Mass Spectrometry-Based Characterization of the Erythromycin-Induced Ribosome