Structure and Function of the Escherichia coliRNA-Binding Global Regulatory Protein CsrA Público
Mercante, Jeffrey Wade (2009)
Abstract
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 (1, residues 2-7) of the protein while region 2 was situated close to the C-terminus (5, 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 1 from one subunit positioned adjacent and parallel to 5 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.
Table of Contents
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 alpha-helical proteins ......................................................................43 alpha/beta protein domains .......................................................................45 Zinc finger domain ..........................................................................47
Multimeric RNA-binding motifs
...................................................48 The CsrA motif ................................................................................49 References..............................................................................................................51
Chapter 2. Comprehensive Alanine-scanning Mutagenesis of Escherichia coli CsrA Defines Two Subdomains of Critical Functional Importance ...................................................................................................................88 Summary ................................................................................................................89 Abstract ..................................................................................................................90 Introduction............................................................................................................91 Experimental procedures .......................................................................................94 Results....................................................................................................................99 Discussion ............................................................................................................106 Acknowledgements..............................................................................................112 References............................................................................................................112
Chapter 3. Molecular geometry of CsrA (RsmA) binding to RNA and its implications for regulated expression ...............................................................138 Summary ..............................................................................................................139 Abstract ................................................................................................................140 Introduction..........................................................................................................141 Results..................................................................................................................144 Discussion ............................................................................................................156 Materials and Methods.........................................................................................164 Acknowledgements..............................................................................................170 References............................................................................................................170
Chapter 4. Discussion .............................................................................................196 CsrA bilateral symmetry and amino acid conservation ......................................196 CsrA molecular geometry and target site binding ..............................................202 Outlook ................................................................................................................205 References............................................................................................................206
Appendix. Additional Publications ...................................................................213 CsrA Inhibits Translation Initiation of Escherichia coli hfq by Binding to a Single Site Overlapping the Shine-Dalgarno Sequence .....................................213 The evolution of contact-dependent inhibition in non-growing populations of Escherichia coli ......................................................................................................250
LIST OF TABLES AND FIGURES Chapter 1.
Fig. 1-1. Outline of the the E. coli K12 Csr circuitry .......................................................87
Chapter 2. Table 2-1. Bacterial strains, plasmids and bacteriophage used in this study ..................124 Table 2-2. Oligonucleotide primers used in this study .........................................................125 Fig. 2-1. Phenotypic and regulatory effects of CsrA alanine replacement mutations ..............132 Fig. 2-2. Quantitative immunoblotting of TRMG1655 ( csrA::kan) expressing wild type and 58 alanine-scanning mutant CsrA proteins ...............................................................................133 Fig. 2-3. Gel mobility shift assays for the binding of wild type and mutant CsrA proteins to a 16 nucleotide high-affinity RNA target ....................................................................................134 Fig. 2-4. Correlation between the " in vivo regulatory defect" (Fig. 2-6) and the equilibrium- binding constant ( Kd) (Fig. 2-3) of 8 CsrA mutant proteins ...................................................135 Fig. 2-5. Critical residues of CsrA mapped onto the 3-D model of Yersinia enterocolitica CsrA. ..........................................................................................................................................136 Fig. 2-6. Comparison of CsrA orthologs from 6 Phyla, highlighting regions that were crucial for RNA binding and in vivo regulation with predicted amino acid functions ..............................137
Chapter 3. Table 3-1. Synthetic RNA oligonucleotides used in this study ......................................181 Table 3-2. Bacterial strains and plasmids used in this study ..........................................182 Table 3-3. RNA targets of CsrA (or its orthologs) and predicted or experimentally determined spacing .............................................................................................................................183 Fig. 3-1. Electrophoretic Mobility Shift Assay (EMSA) demonstrating that WT-CsrA contains 2 independent RNA binding surfaces while the HD-CsrA contains only one .188 Fig. 3-2. EMSA demonstration that HD-CsrA binds to a single-site RNA with approximately one third the avidity of WT-CsrA ...........................................................189 Fig. 3-3. EMSA analysis of the optimal CsrA binding site spacing using a series of synthetic RNA oligonucleotides ............................................................................. 190-191 Fig. 3-4. Stoichiometric measurements of CsrA:RNA ratios within shifted complexes 192 Fig. 3-5. Effects of WT-CsrA or HD-CsrA on the expression of glgC'-`lacZ or glgCGGA' -`lacZ translational fusions in S-30 coupled transcription-translation reactions. .........................................................................................................................193 Fig. 3-6. Boundary Analysis revealing additional uncharacterized CsrA target sites in the glgCAP 5'-leader ............................................................................................................194 Fig. 3-7. A model for CsrA regulation by binding to the 5' leader of repressed transcripts, based on results from RNA gel shifts and glgC S30 transcription-translation ...............195
Chapter 4.
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