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Title: Kinetic studies of DNA-dependent RNA polymerase from E. coli
Author: Roach, Peter John
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1972
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DNA-dependent RNA polymerase was isolated in a highly purified form from E. coli MRE 600 by the method previously established in this laboratory. The polypeptide composition of enzyme samples was routinely analysed by SDS-polyacrylamide gel electrophoresis. This technique gave not only on analysis of the purity of RNA polymerase samples but could also yield, in some instances, estimates for mol.wt.'s of polypeptides present. RNA polymerase prepared in this laboratory had always contained a polypeptide, theta , in relatively high concentration. The separation of theta from RNA polymerase, by glycerol gradient centrifugation, is reported here. It was found also that theta existed as an, aggregate of the 58 000 mol.wt. monomer observed by SDS-polyacrylamide gel electrophoresis, during glycerol gradient centrifugation at high ionic strength. The properties of theta resemble those of a protein with ATPase activity reported very recently by Paetkau & Coy (1972). By use of the SDS-polyacrylamide gel electrophoresis technique, the preciso polypeptide composition of RNA polymerase preparations was found, in general, to be dependent on the purification procedure used. A fluorimetric assay for DNA directed RNA polymerase activity was developed. This method is based on the fluorescence properties of ethidium bromide-nucleic acid complexes and had been described originally for measuring umprimed RNA polymerase activity. A variation of the fluorimetric assay was employed to measure polynucleotide phosphorylase activity, which was present in some RNA polymerase samples. The dependence of RNA polymerase activity on ionic conditions was investigated, in order to establish conditions for subsequent kinetic experiments. The time course of RNA synthesis by RNA polymerase was studied and, in particular, the RNA product was considered as an inhibitor of the reaction. A complex inhibition pattern was observed, which did, however, conform approximately to non-competitive inhibition at high substrate concentration. Kinetic experiments were carried out using T7 DNA and calf thymus DNA templates. The kinetic behaviour of RNA polymerase was dependent on various factors; the nature of the template, the presence of the initiation factor, and whether purine or pyrimidine nucleotides were varied. In order to use a better defined enzyme-template system, the kinetic properties of RNA polymerase core enzyme (apparently the simplest active form of the enzyme) with a poly(dA-dT) template were investigated. With this system, the steady state rate equation for a two substrate ping pong mechanism, with substrate inhibition by ATP, was found to be applicable over a certain range of substrate concentrations. At higher substrate concentrations, deviations from this rate law were observed. Model rate equations, of the general form of steady state rate law were analysed, by the least squares method, for their ability to fit the experimental data. The form of the equation that best described the experimental results could be explained in several ways; one of these was the existence of alternative reaction pathways. This eventuality was investigated further by analysing the poly(A-U) product in various ways : nearest neighbour analysis, determination of the 5'-terminal, and the adjacent, base residues, and measurement of the gross base stoichiometry. The results of these experiments provided some evidence for the operation of alternative reaction pathways at the stage of the initiation of poly(A-U) synthesis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available