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Downloaded From Distribution Agreement In presenting this thesis or dissertation as a partial fulfillment of the requirements for an advanced degree from Emory University, I hereby grant to Emory University and its agents the non-exclusive license to archive, make accessible, and display my thesis or dissertation in whole or in part in all forms of media, now or hereafter known, including display on the world wide web. I understand that I may select some access restrictions as part of the online submission of this thesis or dissertation. I retain all ownership rights to the copyright of the thesis or dissertation. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. Signature: ______________________________ __________ Jeffrey W. Mercante Date Graduate Division of Biological and Biomedical Sciences Program in Microbiology and Molecular Genetics Structure and Function of the Escherichia coli RNA-Binding Global Regulatory Protein CsrA By Jeffrey W. Mercante Doctor of Philosophy Graduate Division of Biological and Biomedical Sciences Program in Microbiology and Molecular Genetics ___________________________ Tony Romeo Advisor ___________________________ ___________________________ Charles P. Moran William Shafer Committee Member Committee Member ___________________________ ___________________________ Daniel Kalman Philip N. Rather Committee Member Committee Member Accepted: ___________________________ Lisa A. Tedesco, Ph.D. Dean of the Graduate School ___________________________ Date Structure and Function of the Escherichia coli RNA-Binding Global Regulatory Protein CsrA By Jeffrey W. Mercante B.A., Southern Methodist University, 1998 M.S., Louisiana State University, 2002 Advisor: Tony Romeo, Ph.D. An Abstract of A dissertation submitted to the Faculty of the Graduate School of Emory University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Division of Biological and Biomedical Sciences Program in Microbiology and Molecular Genetics 2009 ABSTRACT Structure and Function of the Escherichia coli RNA-Binding Global Regulatory Protein CsrA By Jeffrey W. Mercante In Escherichia coli K12, the small RNA-binding protein, CsrA, is the central component of a pathway that has pleiotropic effects on cellular processes such as carbon metabolism, motility and biofilm formation. CsrA binds to the 5’-untranslated leader of mRNA transcripts and, in repressed messages, inhibits ribosome access and thus translation. At the outset of this project, little was known about the structure of CsrA except that it was a homodimer. Therefore, a comprehensive alanine-scanning mutagenesis of the protein was undertaken to determine which amino acids were important for RNA- binding and regulation of gene expression. Two regions of the protein were identified where mutations caused a dramatic affect on CsrA activity. Region 1 was found at the N-terminus (β1, residues 2-7) of the protein while region 2 was situated close to the C-terminus (β5, residues 40-47). When these regions were mapped to a recently determined 3D CsrA structure two RNA-binding subdomains were defined on opposite sides of the bilaterally symmetrical protein; each subdomain consisted of β1 from one subunit positioned adjacent and parallel to β5 from the other subunit in three-dimensional space. Critical amino acids that make up the binding surfaces, including the most important residue, R44, were found to be highly conserved in all species examined. Construction of a heterodimer CsrA protein (HD-CsrA) that contains only a single normally functioning RNA-binding surface and EMSA studies revealed that CsrA has the capacity to bind one or two independent RNA molecules. Furthermore, CsrA can interact with two RNA target sites within the same RNA oligonucleotide simultaneously (dual binding). A distance of 18 nucleotides was established as optimal spacing between targets to allow for dual binding. Finally, a native E. coli CsrA target that contains multiple binding sites, glgCAP, was repressed ~14-fold more efficiently in vitro from the wild-type CsrA dimer than by HD-CsrA. When one CsrA target site was deleted in the glgCAP leader, repression by wild-type CsrA decreased while HD-CsrA regulation was unchanged, thus establishing that dual binding has biological relevance. Structure and Function of the Escherichia coli RNA-Binding Global Regulatory Protein CsrA By Jeffrey W. Mercante B.A., Southern Methodist University, 1998 M.S., Louisiana State University, 2002 Advisor: Tony Romeo, Ph.D. A dissertation submitted to the Faculty of the Graduate School of Emory University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Division of Biological and Biomedical Sciences Program in Microbiology and Molecular Genetics 2009 ACKNOWLEDGEMENTS I am grateful for the support and encouragement of many people without whom this journey would not have been possible. First I want to thank my mentor, Tony Romeo, who has been a steady voice of reason, wisdom and encouragement through these years. His analytical mind and unsinkable optimism guided me through many less- than-successful experiments and he taught me to wade through and distill the most important aspects of my research into a coherent, original body of work. I may never learn to write as succinctly as him, but at least I know now to what I should aspire. I want to thank my dissertation committee – Drs. Charles Moran, William Shafer, Daniel Kalman and Philip Rather - for the time and effort they have put forth and the commitment they have made to my graduate training. Thank you all for setting high standards for me and my work. I am grateful to the members of the Romeo Lab, both past and present, including Kazushi “Hands of Gold” Suzuki, Yoshi “Where’s the Beer” Itoh, Amy “Tennis shoe allergy” Kirby and Adrianne “I Brake for Pandas” Edwards. I especially want to thank Carlos “Bling-Bling ‘Los’” Goller who spent long hours in lab with me trying to figure out the true meaning of good science. Your company and council are sorely missed. I want to also thank Keith Wilkinson and Monica Taylor for the opportunity to serve on the DSAC committee representing the Microbiology and Molecular Genetics Program. I truly enjoyed dabbling in the politics and boardroom discussions that make the GDBBS what it is. I am grateful for my loving, loyal family, including my mother Josie, and my father Francis who always ask about my studies and who sit uncomplainingly as I explain my ongoing projects in painful detail. I truly appreciate their undying support and patience. And last, but certainly not least, I thank my wife, Alex, who has sustained me through my studies and late nights even as she has worked to succeed in her own projects. I could not have done this without you. You are my counselor, confidant, and best friend. Thank you for standing with me as I trudged through the hard times. I wish you luck in your degree and I want you to know that I will always be there to support you in your endeavors, I love you with all my heart. TABLE OF CONTENTS Chapter 1. Introduction..............................................................................................1 Rationale and specific aims .....................................................................................1 Background and significance...................................................................................2 Post-transcriptional processes modulated by RNA-binding proteins .........4 Eukaryotic RBPs important for post-transcriptional regulation..4 Splicing ................................................................................4 Nuclear Export.....................................................................5 Translation initiation............................................................5 Translation termination and decay.......................................7 Prokaryotic RBPs important for post-transcriptional regulation.9 Ribosomal and ribosome-associated RBPs .........................9 Non-ribosomal RBPs ........................................................11 Cold shock proteins ...............................................11 AcnA and AcnB aconitases ...................................12 Pseudomonas Crc .................................................13 Lactococcus group II Intron maturase, LtrA ........14 Bacteriophage coat proteins .................................14 Phage T4 autoregulatory proteins ........................15 B. subtilis Tryptophan regulation and TRAP ........16 CsrA and the E. coli K12 Csr system-Overview ...17 Noncoding sRNAs CsrB and CsrC.........18 BarA/UvrY two-component system .......19 The RNA-binding protein CsrA ............21 CsrD protein and CsrB/C decay ............22 CsrA-regulated genes in E. coli K12 ......23 Csr/Rsm Circuitry in other bacteria .....................28 Salmonella enterica serovar Typhimurium ..........................................28 Legionella pneumophila ..........................31 Vibrio spp. .................................................34 Erwinia spp. ..............................................35 Pseudomonas spp......................................36 CsrA in various other bacteria ..............38 Protein RNA-binding motif families ..........................................................41 Small basic arginine-rich motifs (ARM)........................................41 All α-helical proteins ......................................................................43 α/β protein domains .......................................................................45
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