Off-campus UNL users: To download campus access dissertations, please use the following link to log into our proxy server with your NU ID and password. When you are done browsing please remember to return to this page and log out.
Non-UNL users: Please talk to your librarian about requesting this dissertation through interlibrary loan.
Studies of primase from Escherichia coli with its substrates and effectors
Abstract
Escherichia coli primase is an essential DNA-dependent RNA polymerase that initiates DNA replication. Primase must interact with a single-stranded DNA template, ribonucleotides, zinc and magnesium to carry out its function. In vivo, the main role of primase is to synthesize a RNA primer that serves as the initiating polymer for Okazaki fragment synthesis on the lagging strand. The work described in this dissertation describes the binding affinity of primase for some of its substrates. Determination of the affinity of primase for its ssDNA template was attempted using two different biophysical techniques. First, an affinity column with immobilized primase or ssDNA template was developed. Secondly, a double filter binding assay was used to quantitate the amount of ssDNA bound to protein. Both of these methods were only partially successful in measuring the weak primase-template interaction. In a study of primase conformation, it was found that primase was markedly influenced by high concentrations of magnesium acetate. High magnesium acetate shifted a major trypsin cleavage site from residue 110 to residues 34 and 56. The high magnesium acetate conformation was found to be inactive in both the coupled RNA/DNA synthesis assay and the simple primer synthesis assay. A study of the inhibition mechanism of a fluorescent nucleotide analog, $\gamma$-aminonapthalene sulfonate uridine 5$\sp\prime$-triphosphate (AmNS-UTP), revealed the presence of a nucleotide modulation site on primase. Not only AmNS-UTP, but high ATP, UTP, and CTP also bound to the modulation site to decrease the activity by 30-50%. The nucleotide modulation site was distinct from the initiation and polymerization nucleotide sites. The nucleotide modulation effect did not require that the nucleotide be incorporated into the RNA polymers being synthesized. At cellular nucleotide concentrations, the activity of primase would be expected to be maximally inhibited.
Subject Area
Biochemistry|Biophysics
Recommended Citation
Urlacher, Teresa Mae, "Studies of primase from Escherichia coli with its substrates and effectors" (1996). ETD collection for University of Nebraska-Lincoln. AAI9715989.
https://digitalcommons.unl.edu/dissertations/AAI9715989