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<p> Sigma S (RpoS), encoded by rpoS, is a subunit of RNA polymerase holoenzyme that controls the expression of many genes in stationary phase of various gram negative bacteria. Escherichia coli expresses these genes to withstand environmental stress and nutrient starvation. Several naturally occurring mutant alleles of the gene have been reported and indicate key differences from laboratory strains. We sought to explore the role of natural alleles of the rpoS gene (from non- K12 strains) and thus the sigma subunit relative to the K12 allele. To study the effect of the rpoS polymorphism on gene expression ofRpoS regulon members, rpoS alleles from ECOR- 21, ECOR-28, ECOR-37 and ECOR-40 as well as MG1655 were cloned into the same background, MG1655 ΔrpoS:cat osmY-lacZ. Sequence analysis showed rpoS alleles from all the natural strains tested were different from MG1655 and each other. The strain with rpoS allele from ECOR-28 had increased expression of osmY and katE similar to MG1655. In contrast, rpoS allele from ECOR- 37 showed low expression of osmYbut not as low as ECOR-21 and ECOR-40 which had expression similar to the rpoS mutant. Not surprisingly, recombinant strains with rpoS alleles from ECOR-21, ECOR-37 and ECOR-40 showed no expression of katE (HPII). These suggest that RpoS in ECOR-28 has high activity similar to wildtype K12 strain while RpoS in ECOR-21, ECOR-37 and ECOR-40 has very low or no activity. We conclude that natural E. coli strains have polymorphism in their rpoS ORF which cause variation in the regulatory activities of RpoS on its regulon. </p> / Thesis / Master of Science (MSc)

The human mitochondrial DNA polymerase (Pol γ) catalyzes mitochondrial DNA synthesis, and thus is essential for the integrity of the organelle. Mutations of Pol γ have been implicated in more than 150 human diseases. Reduced Pol γ activity caused by inhibition of anti-HIV drugs targeted to HIV reverse transcriptase confers major drug toxicity. To illustrate the structural basis for mtDNA replication and facilitate rational design of antiviral drugs, I have determined the crystal structure of human Pol γ holoenzyme. The structure reveals heterotrimer architecture of Pol γ holoenzyme with a monomeric catalytic subunit Pol γA, and a dimeric processivity factor Pol γB. While the polymerase and exonuclease domains in Pol γA present high structural homology with the other members of the DNA Pol I family, the spacer between the two functional domains shows a unique fold, and constitutes the subunit interface. The structure suggests a novel mechanism for Pol γ’s high processivity of DNA replication. Furthermore, the structure reveals dissimilarity in the active sites between Pol γ and HIV RT, thereby indicating an exploitable space for design of less toxic anti-HIV drugs. Interestingly, the structure shows an asymmetric subunit interaction, that is, one monomer of dimeric Pol γB primarily participates in interactions with Pol γA. To understand the roles of each Pol γB monomer, I generated a monomeric human Pol γB variant by disrupting the dimeric interface of the subunit. Comparative studies of this variant and dimeric wild-type Pol γB reveal that each monomer in the dimeric Pol γB makes a distinct contribution to processivity: one monomer (proximal to Pol γA) increases DNA binding affinity whereas the other monomer (distal to Pol γA) enhances the rate of polymerization. The pol γ holoenzyme structure also gives a rationale to establish the genotypic-phenotypic relationship of many disease-implicated mutations, especially for those located outside of the conserved pol or exo domains. Using the structure as a guide, I characterized a substitution of Pol γA residue R232 that is located at the subunit interface but far from either active sites. Kinetic analyses reveal that the mutation has no effect on intrinsic Pol γA activity, but shows functional defects in the holoenzyme, including decreased polymerase activity and increased exonuclease activity, as well as reduced discrimination between mismatched and corrected base pair. Results provide a molecular rationale for the Pol γA-R232 substitution mediated mitochondrial diseases. / text

A method is described for the rapid purification of RNA polymerase holoenzyme from small amounts of Escherichia coli cells. Chromatography of a crude extract on a single-stranded DNA agarose column followed by gell filtration chromatography gave 95% pure holoenzyme. The enzyme kinetic characteristics on T7 DNA identical to those of RNA polymerase purified by other more laborious procedures.