The characterization of Escherichia coli 4.5S RNA and the importance of its highly conserved residues for protein localization and protein synthesis

Abstract

CHAPTER 1. General Introduction Thesis organization Introduction Characterization of the signal recognition particle pathway Discovery and characterization of the SRP SRP implicated in bacteria Early characterization of 4.5S RNA 4.5S RNA implicated in protein synthesis "Sequence gazing": 4.5S RNA implicated in protein localization SRP in E. coli 4.5S RNA in protein synthesis continued Function of the SRP in E. coli Structural analysis of 4.5S RNA Mutational analysis of 4.5S RNA Reference CHAPTER 2. Characterization of highly conserved bases of Escherichia coli 4.5S RNA through the construction of new F′ ′ ′ ′ factors ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCE CHAPTER 3. The role of Escherichia coli 4.5S RNA in protein synthesis is not essential ABSTRACT INTRODUCTION REFERENCE CHAPTER 4. New pSC101-derivative cloning vectors with elevated copy numbers ABSTRACT INTRODUCTION iii MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCE CHAPTER 5. New methods for rapid depletion of 4.5S RNA in Escherichia coli ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCE CHAPTER 6. General Conclusions Reference vi ABSTRACT 4.5S RNA, the product of the essential ffs gene of Escherichia coli, is a highly conserved, 114-base molecule that functions as a component of the signal recognition particle (SRP) and also has been implicated in protein synthesis. To address conflicting results reported in other studies and determine the essential function of 4.5S RNA, we have developed an improved genetic system to characterize ffs mutants. Multiple ffs alleles altered at several highly conserved positions were constructed. To test the ability of the alleles to complement an ffs knockout mutation in single copy, a method to transfer the genes to F' factors with minimal manipulation was developed. These F' factors along with plasmids that express ffs in multiple copy number were used to assess the ability of mutant 4.5S RNA to function in vivo. Despite the high degree of evolutionary conservation, only a single base (C62) was indispensable for RNA function under all conditions tested. To determine the importance of these bases for the interaction between 4.5S RNA and either Ffh or EF-G, an assay to measure the interaction of Ffh or EF-G with mutant 4.5S RNA molecules in vivo was also developed. Defects in Ffh binding correlated with loss of SRP-dependent protein localization and loss of viability. Defects in EF-G binding were found to correlate with a slight decrease in protein synthesis but did not correlate with loss of viability. These results clarify inconsistencies from prior studies and further support that 4.5S RNA is essential only as a component of the SRP. These results also yielded convenient methods to study the function of multiple alleles and to qualitatively measure an interaction between a protein and an RNA. 1 CHAPTER 1. General Introduction Thesis organization This thesis is structured into six chapters including: this first chapter containing the introduction and literat...