Biophysical Society Thematic Meeting| Lima 2019

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

Poster Abstracts

16-POS Board 16 TRANSCRIPTIONAL DYNAMIC OF MYCOBACTERIUM TUBERCULOSIS RNA POLYMERASE Keren A Espinoza Huertas 1 ; Daniel Guerra Giraldez 1 ; Omar Herrera Asmat 2 ; 1 Universidad Peruana Cayetano Heredia, Science faculty, Lima, Peru 2 University of California, Berkeley, CA, USA Mycobacterium tuberculosis (Mtb) is an infectious agent that causes more than one million of annual deaths worldwide. RNA polymerase (RNAP) is one of the most important therapeutic targets to fight Mtb, therefore a detailed characterization of this protein and its activity is necessary to understand the mechanisms of antibiotic resistance and contribute to the development of new treatments against this bacterium. Given the slow growth of Mtb, its regulatory mechanisms are probably very different from what is observed in the currently most well-known transcription systems (mainly E. coli and Archaea). Using optical tweezers, we have manipulated individual molecules of Mtb-RNAP in order to evaluate its activity during transcription elongation. Elongation activity of Mtb-RNAP was evaluated with saturant concentrations of NTPs at 20°C. We obtained 13 traces at 5pN and 5 traces at 15pN. A total velocity of 5.78±2.17 nt/s at 5pN and a velocity of 2.53±0.78 nt/s at 15pN were calculated. The free pause velocity (instant rate of transcription) at 5pN was 6.48 ± 0.40 nt/s, and, it was 7.13 ± 0.39 nt/s at 15 pN. There is no significant difference between velocities at different forces; however, the free pause velocity of Mtb is significantly lower than the E. coli velocity (±14 nt/s). We report, for the first time, a description of the Mtb RNAP’s dynamics. The average speed as number of nucleotides incorporated per second, the distribution of pause times, the pause-free velocity, and the force dependence of these variables are reported too. These results bring the first clues about the differences in transcriptional dynamic between Mtb and E. coli. Moreover, this novel biochemical approach will allow subsequent studies in the regulation of gene expression, interaction with inhibitors and the functional consequences of polymorphisms associated with antibiotic resistance.

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