Mutations that increase the copy number of the pSC101 replicon have been used for construction of new cloning vectors. Replacement of glutamate at position 93 in RepA yields plasmids that replicate at medium (27 copies/cell) and high (∼240 copies/cell) copy numbers. Based on the crystal structure of RepE, a structurally similar replication initiator protein from the F factor, the pSC101 repA mutants are predicted to be defective in dimerization. The cloning vectors permit increased expression of gene products along with the advantages of pSC101-derivative plasmids, including stable maintenance and compatibility with ColE1 plasmids. The plasmids also allow blue/white screening for DNA inserts and impart resistance to ampicillin, chloramphenicol and kanamycin. The vectors were used in a genetic assay to suppress temperature-sensitive mutants of ffh, encoding the protein component of the E. coli signal recognition particle, by overproduction of 4.5S RNA. While expression of 4.5S RNA from a wild type pSC101-derivative plasmid was not sufficient for suppression, use of the new vectors did suppress the temperature-sensitive phenotype.

To more clearly understand the function of conserved bases of 4.5S RNA, the product of the essential ffs gene of Escherichia coli, and to address conflicting results reported in other studies, we have developed a new genetic system to characterize ffs mutants. Multiple ffs alleles were generated by altering positions that correspond to the region of the RNA molecule that interacts directly with Ffh in assembly of the signal recognition particle. To facilitate characterization of the ffs mutations with minimal manipulation, recombineering was used to construct new F factors to easily move each allele into different genetic backgrounds for expression in single copy. In combination with plasmids that expressed ffs in multiple copy numbers, the F factors provided an accurate assessment of the ability of the different 4.5S RNA mutants to function in vivo. Consistent with structural analysis of the signal recognition particle (SRP), highly conserved bases in 4.5S RNA are important for binding Ffh. Despite the high degree of conservation, however, only a single base (C62) was indispensable for RNA function under all conditions tested. To quantify the interaction between 4.5S RNA and Ffh, an assay was developed to measure the ability of mutant 4.5S RNA molecules to copurify with Ffh. Defects in Ffh binding correlated with loss of SRP-dependent protein localization. Real-time quantitative PCR was also used to measure the levels of wild-type and mutant 4.5S RNA expressed in vivo. These results clarify inconsistencies from prior studies and yielded a convenient method to study the function of multiple alleles.

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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...

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