Physico-chemical aspects of E.coli MRE 600 DNA-dependent RNA polymerase

Lochhead, Douglas Stewart (1972) Physico-chemical aspects of E.coli MRE 600 DNA-dependent RNA polymerase. PhD thesis, University of Glasgow.

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In the introduction an outline of the currently known physical, chemical and enzymic properties of E. coli DNA-dependent RNA polymerase (E.C. is given. Some aspects of the regulation and control of RNA synthesis in E. coli with special reference to specific protein factors are mentioned. Two purification procedures for the preparation and purification of RNA polymerase from E. coli MRE 600 are described, compared and discussed. The subunit composition of E. coli MRE 600 RNA polymerase on analysis by SDS-polyacrylamide gel electrophoresis shows the presence of five major protein species already described in the literature, and a fifth subunit of molecular weight 58,000 daltons is shown to be strongly associated with E, coli MRE 600 RNA polymerase and it behaves in a physicochemical manner similar to the O subunit except that it has no C-like activity determined by its inability to stimulate RNA synthesis on a T7 DEA template with core enzyme. A crude fraction has been isolated from E. coli MRE 600 containing a protein factor which has the property of inhibiting both the initiation and rate of RNA synthesis by RNA polymerase holoenzyrae in vitro. The inhibitor protein has a molecular weight of 320,000 daltons and has associated with it some proteolytic activity. The amino acid composition of E. coli MRE 600 RNA polymerase, core enzyme and holoenzyme, is presented. The ultra-violet spectrum of RNA polymerase has a maximum at 275.5nm and e. minimum at 251nra. The enzyme has e, specific absorbance of 0.63/mg/ml, A standard curve was prepared from the spectra of E, coli MRE 600 DM and RNA polymerase to allow the calculation of nucleic acid contamination of the various fractions during the enzyme preparation. A formula was derived for the spectrophotometric determination of the concentration of RNA polymerase in the presence of any contaminating nucleic acid. The ORD of native RNA polymerase is that of a typical protein containing right-handed helical and random coil (disordered) conformations. The helix content of RNA polymerase is around 32%. The presence of other conformations is also indicated. The conformation of E, coli MRE 600 RNA polymerase shows very little change with increasing ionic strength although the enzyme dissociates from the dimer to the monomer over the range of ionic strengths studied. The conformation of RNA polymerase in various denaturing solutions (BM-urea, 1% SDS and 6M-GUHC1) was also determined. There is a decrease in the [m']3232 polymerase holoenzyme when it is bound to DNA indicating a conformational change in the enzyme. There is no detectable denaturation of the DNA. Thermal ORD studies indicate that RNA polymerase is unstable when bound to DNA. This thermal denaturation is irreversible. The possible origin of the 0 subunit as a breakdown product of the 0 subunit of RNA polymerase holoenzyme, its role in the regulation of RNA synthesis and transcription, and the properties and function of the RNA polymerase inhibitor protein are discussed. The physical and chemical properties of the E. coli MRS 600 RNA polymerase are compared with those of other strains of E. coli.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: A FH Anderson
Keywords: Biochemistry
Date of Award: 1972
Depositing User: Enlighten Team
Unique ID: glathesis:1972-73827
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Jun 2019 08:56
Last Modified: 14 Jun 2019 08:56

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