Insights into the Co-Evolution of Ribosomal Protein S15 with its Regulatory RNAs Author: Betty L. Slinger Persistent link: http://hdl.handle.net/2345/bc-ir:106793 This work is posted on eScholarship@BC, Boston College University Libraries. Boston College Electronic Thesis or Dissertation, 2016 Copyright is held by the author, with all rights reserved, unless otherwise noted. Boston College The Graduate School of Arts and Sciences Department of Biology INSIGHTS INTO THE CO-EVOLUTION OF RIBOSOMAL PROTEIN S15 WITH ITS REGULATORY RNAS a dissertation by BETTY L. SLINGER submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy May 2016 © copyright by BETTY LOU SLINGER 2016 BOSTON COLLEGE Graduate School of Arts & Sciences The Dissertation of: ____Betty L. Slinger____ (Student’s Name) Title: INSIGHTS INTO THE CO-EVOLUTION OF RIBOSOMAL PROTEIN S15 WITH ITS REGULATORY RNAS________ Submitted to the Department of: ___BIOLOGY_______________________ In partial fulfillment of the requirements for the degree of: ______________Doctor of Philosophy_______________________ In the Graduate School of Arts & Sciences has been read and approved by the Committee: Welkin Johnson__________________________________________________________ Chair Thomas Chiles___________________________________________________________ Member Eric Folker______________________________________________________________ Member Charles Hoffman_________________________________________________________ Member Michelle. M. Meyer_______________________________________________________ Advisor, Member May 23, 2016_______________________________ Date ABSTRACT INSIGHTS INTO THE CO-EVOLUTION OF RIBOSOMAL PROTEIN S15 WITH ITS REGULATORY RNAS Betty L. Slinger Thesis Advisor: Michelle M. Meyer Ribosomes play a vital role in all cellular life translating the genetic code into functional proteins. This pivotal function is derived from its structure. The large and small subunits of the ribosome consist of 3 ribosomal RNA strands and over 50 individual ribosomal proteins that come together in a highly coordinated manner. There are striking differences between eukaryotic and prokaryotic ribosomes and many of the most potent antibacterial drugs target bacterial ribosomes (e.g. tetracycline and kanamycin). Bacteria spend a large amount of energy and nutrients on the production and maintenance of these molecular machines: during exponential growth as much as 40% of dry bacterial mass is ribosomes (Harvey 1970). Because of this, bacteria have evolved an elegant negative feedback mechanism for the regulation of their ribosomal proteins, known as autoregulation. When excess ribosomal protein is produced, unneeded for ribosome assembly, the protein binds a structured portion of its own mRNA transcript to prevent further expression of that operon. Autoregulation facilitates a quick response to changing environmental conditions and ensures economical use of nutrients. My thesis has investigated the autoregulatory function of ribosomal protein S15 in diverse bacterial phyla. In many bacterial species, when there is excess S15 the protein interacts with an RNA structure formed in the 5’-UTR of its own mRNA transcript that I enables autoregulation of the S15-encoding operon, rpsO. For many ribosomal proteins (ex. L1, L20, S2) there is striking homology and often mimicry between the recognition motifs within the rRNA and the regulatory mRNA structure. However, this is not the case for S15-three different regulatory RNA structures have been previously described in E. coli, G. stearothermophilus, and T. thermophilus (Portier 1990, Scott 2001, Serganov 2003). These RNAs share little to no structural homology to one another, nor the rRNA, and they are narrowly distributed to their respective bacterial phyla, Gammaproteobacteria, Firmicutes, and Thermales. It is unknown which regulatory RNA structures control the expression of S15 outside of these phyla. Additionally, previous work has shown the S15 homolog from G. stearothermophilus is unable to regulate expression using the mRNA from E. coli. These observations formulate the crux of the question this thesis work endeavors to answer: What drove the evolution of such diverse regulatory RNA structures in these different bacteria? In Chapter II, “Discover and Validate Novel Regulatory Structures for Ribosomal Protein S15in Diverse Bacterial Phyla”, I present evidence for the in silico identification of three novel regulatory RNA structures for S15 and present experimental evidence that one of these novel structures is distinct from those previously described. In Chapter III, “Co-evolution of Ribosomal Protein S15 with Diverse Regulatory RNA Structures”, I present evidence that the amino acid differences in S15 homologs contribute to differences in mRNA binding profiles, and likely lead to the development of the structurally diverse array of the regulatory RNAs we observe in diverse bacterial phyla. In Chapter IV, “Synthetic cis-regulatory RNAs for Ribosomal Protein S15”, I investigate the derivation of novel cis-regulatory RNAs for S15 and find novel structures are readily- II derived, yet interact with the rRNA-binding face of S15. Together the work presented in this thesis advances our understanding of the co-evolution between ribosomal protein S15 and its regulatory RNAs in diverse bacterial phyla. III DEDICATION To my Husband, Spencer, For Everything, Forever! IV ACKNOWLEDGEMENTS There are a number of people without whom this dissertation would not be possible and to whom I am greatly indebted. I would like to express deepest gratitude to Michelle M. Meyer, my mentor and PhD thesis advisor, who expertly guided me through my six years of graduate education at Boston College and who shared the excitement of five years of discovery. It was through her constant support, generous advice, and incredible patience that performing all of my experimental work, as well as publishing it and presenting it at scientific conferences was possible. My appreciation also extends to my laboratory colleagues, especially Shermin Pei and Arianne Babina, for your constant encouragement, support and making our lab a positive, good-humored, and intellectually-stimulating environment to perform research. Thank you to my thesis committee members, Tom Chiles, Welkin Johnson, Eric Folker, and Charlie Hoffman for your invaluable advice and guidance. Thank you to my fellow graduate students and friends who make this journey through grad school about the journey, and not the destination. Thanks for all the Thirsty Thursdays, running 5K’s with me for the beer tent, and all the game nights! I am indebted to my parents, Ron and Elsie Slinger, and sisters, Mary and Laura, for their unequivocal support and love. And finally, I acknowledge my husband, Spencer, who is my champion and who has blessed me with a life of joy and love in the hours when lab lights were off. V TABLE OF CONTENTS ABSTRACT ......................................................................................................................... i DEDICATION ................................................................................................................... iv ACKNOWLEDGEMENTS ................................................................................................ v TABLE OF CONTENTS ................................................................................................... vi LIST OF FIGURES ........................................................................................................... xi DATASETS ..................................................................................................................... xiv CHAPTER I The Role of Ribosomal Protein S15 in the Prokaryotic Ribosome ................................ 2 Cis-acting RNA Elements Regulate Gene Expression in Bacteria ................................. 6 Evolution of RNA-Protein Regulatory Interactions ..................................................... 10 The S15-mRNA Interaction in Bacteria ....................................................................... 14 FIGURES & LEGENDS............................................................................................... 19 Figure 1.1 Complexity of The Prokaryotic Ribosome .............................................. 19 Figure 1.2 S15-rRNA Binding Interaction................................................................ 20 Figure 1.3 Conservation of Residues and Nucleotides Involved in S15-rRNA recognition ................................................................................................................ 21 Figure 1.4 Autoregulation of R-Protein S15 ............................................................. 22 Figure 1.5 Phylogenetic Analysis of Regulatory RNAs ........................................... 23 Figure 1.6 rpsO Regulatory RNAs ........................................................................... 24 CHAPTER II INTRODUCTION ........................................................................................................ 26 RESULTS & DISCUSSION......................................................................................... 27 Non-coding Regulatory RNA Discovery Using Comparative Genomics ................ 27 RNAs Identified Are Diverse in Sequence and Secondary Structure ....................... 29 RNA from Alphaproteobacterium Rhizobium radiobacter Specifically Interacts with S15 Protein ...............................................................................................................