Mahurin Honors College Capstone Experience/Thesis Projects

Department

Biology

Document Type

Thesis

Abstract

All bacteria contain a multi-subunit RNA polymerase (RNAPs) that is essential for gene expression. Because of the centrality of these enzymes in cellular life, the structure and function of the various subunits is intensely studied. The primary sequence of the RNAP β’ subunit contains five cysteine residues that are highly conserved. Four of the cysteines coordinate a zinc atom and form the beta prime subunit zinc binding domain (ZBD). Mutation of any one of the ZBD cysteines is lethal to the cell. However, the role of the fifth residue (C58), which is located upstream of the ZBD cysteines, has not been investigated. In previous work, we cloned a copy of the E. coli rpoC onto a plasmid and changed the cysteine at position 58 to an alanine (C58A). Phenotypic analysis suggested that expression of the mutant subunit from the multi-copy plasmid did not support E. coli growth at high temperatures. In this study, we describe the generation of the C58A mutation in single copy on the chromosome using a chromosomal engineering technique. In addition, we investigated if the mutant subunit affects RNA-mediated transcription antitermination.

Advisor(s) or Committee Chair

Rodney King, Ph.D.

Disciplines

Cell Biology | Genetics | Molecular Biology

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