Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Hanna Nune - University of Georgia
Co-Author(s): Jesse Fetcher, Nickolaus Galloway, and Cory Momany, University of Georgia, Athens, GA
RNA polymerases are multi-subunit enzymes that transcribe DNA to RNA. In prokaryotes, the enzyme has five subunits: beta subunit involved in ribonucleotide chain elongation; beta prime subunit that contains the active site for nucleotide chain polymerization; two alpha subunits involved in regulation and DNA recognition; omega subunit; and sigma70 subunit that directs promoter specificity. Although we know a great deal about RNA polymerases, a major question remains: how does RNA polymerase communicate with transcriptional regulators? The objective of this research is to produce RNA polymerase from Acinetobacter baylyi ADP1. The first step of this project was to PCR amplify RNA polymerase genes from ADP1. The PCRamplified DNAs encoding the b and b’ subunits (the rpoB and rpoC genes) were introduced into the vector pRARE2, which has chloramphenicol antibiotic resistance. Each has its own T7 RNA polymerase promoter and terminator. The PCR-amplified DNA encoding a, w, and s subsunits (rpoA, rpoZ, and rpoD) were introduced into the pet-28b vector, which has kanamycin resistance. In this construct, a single T7 promoter is used. The two vectors, pRARE2 and pET-28b are compatible with one another in E. coli, where the proteins will be co-translated. Purified RNA polymerase from ADP1 will allow studies to be performed to examine the communication between RNA polymerase and transcriptional regulators. Ultimately, the enzyme will be used to identify small molecules that prevent transcriptional activation. By blocking essential regulatory networks in microorganisms, the resulting therapeutics would represent a new class of antibiotics.
Funder Acknowledgement(s): National Science Foundation; University of Georgia, College of Pharmacy. CURO Program.
Faculty Advisor: Cory Momany,