Degree Name

PhD (Doctor of Philosophy)

Program

Biomedical Sciences

Date of Award

December 1990

Abstract

A temperature-sensitive mutant of Escherichia coli affected in the alpha subunit of RNA polymerase has been investigated. Gene mapping and complementation experiments placed the mutation to temperature-sensitivity within the alpha operon at 72 min on the bacterial chromosome. The rate of RNA synthesis in vivo and the accumulation of ribosomal RNA were significantly reduced in the mutant at 44$\sp\circ$C. The thermostability at 44$\sp\circ$C of the purified holoenzyme from mutant cells was about 20% of that of the normal enzyme. Assays with T7 DNA as a template showed that the fraction of active enzyme competent for transcription was reduced as a function of assay temperature but that initiation and elongation were not significantly affected by the alpha mutation. A major effect on the fidelity of transcription was observed with the mutant enzyme, with misincorporation on two different templates stimulated about four fold at 37$\sp\circ$C. The role of the alpha dimer in the structure and function of RNA polymerase is discussed. In addition during the course of this study a new procedure for the purification of E. coli RNA polymerase was developed. This method is rapid, convenient, and useful for the preparation of enzyme from 1-5 grams of cells in two days. The ease and speed of this method allowed the rapid characterization of the mutant enzyme. This system should also find application for the purification of small quantities of other bacterial RNA polymerases that share the general chromatographic properties of E. coli RNA polymerase.

Document Type

Dissertation - Open Access

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