Biophysical Society Thematic Meeting| Lima 2019

Revisiting the Central Dogma of Molecular Biology at the Single-Molecule Level

Poster Abstracts

58-POS Board 58 PRODUCTION OF MICROCIN J25 ANTIBACTERIAL PEPTIDE VARIANTS AND ITS ACTIVITY TEST IN THE MYCOBACTERIUM TUBERCULOSIS RNAP. Dahlin Zevallos Aliaga 1 ; Daniel Guerra Giraldez 1 ; 1 UPCH , Biology , Lima, Peru The success of rifampin as anti-tuberculosis therapy indicates that the inhibition of RNA polymerase (RNAP) of Mycobacterium tuberculosis (MtbRNAP) is an effective strategy, however, resistance to this antibiotic is increasing. This is why the finding of new inhibitors for MtbRNAP would be a great contribution to the fight against tuberculosis. Microcin J25 (MccJ25) is an antibiotic peptide whose main molecular target is the RNA polymerase of Escherichia coli (EcoRNAP). Biochemical studies have determined that MccJ25 binds and obstructs the secondary channel of EcoRNAP and have identified the regions of MccJ25 that are responsible for this interaction. They also demonstrated that MccJ25 inhibits RNA polymerases from bacteria phylogenetically related to E.coli, but has no inhibitory activity on the RNAPs of bacteria related to the Mycobacterium genus. Computational analysis indicates that the structure of the secondary channel of EcoRNAP and MtbRNAP is generally conserved but with some amino acid sequence differences that might explain their different MccJ25 susceptibility. Here, I propose to produce variants of MccJ25 through randomly modifying its amino acid sequence in two specific locations that are important for its interaction with residues present EcoRNAP but absent in MtbRNAP. The variants will be produced by PCR- mediated overlap method, and the resulting products will be inserted in a plasmid for the intracellular expression within a reporter bacterium. The reporter bacteria express MtbRNAP, and its activity inside the cell is measured thanks to a GFP gene that is expressed under the control of a promoter recognized by the mycobacterial enzyme. This study intends to serve as prove of principle for the use of a reporter bacterium for high-throughput assays of new inhibitors. Although the variants of MccJ25 to be produced are not intended to serve directly as anti-tuberculosis drugs, their discovery and characterization could contribute to the development of new therapies.

95