Biophysical Society Thematic Meeting| Lima 2019

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

Saturday Speaker Abstracts

DETECTING SINGLE STEPS DURING TRANSCRIPTION AND A HALF- TRANSLOCATED PAUSE COMPLEX OF E. COLI RNA POLYMERASE USING NANOPORE TWEEZERS Ian C Nova 1,2 ; Abhishek Mazumder 3 ; Jonathan M Craig 1 ; Henry Brinkerhoff 1 ; Andrew H Laszlo 1 ; Ian M Derrington 1 ; Matthew T Noakes 1 ; Jesse Huang 1 ; Jonathan W Mount 1 ; Jasmine L 2 University of Washington, Molecular Engineering and Sciences, Seattle, WA, USA 3 Waksman Institute of Microbiology - Rutgers University, New Brunswick, NJ, USA Single-molecule Picometer Resolution Nanopore Tweezers (SPRNT) is a technique that enables observation of single enzyme movement along nucleic acids under an applied force. SPRNT provides measurements of enzyme position along a nucleic acid substrate with sub-Angstrom spatial and millisecond temporal resolution, while simultaneously providing the DNA sequence within the enzyme. We use SPRNT to monitor many E. coli RNA Polymerase (RNAP) core complexes during transcription elongation and pausing with an assisting force. We determine that during elongation at low [NTP], RNAP primarily stalls in a post-translocated state, with brief deviations forward to a hyper-translocated state and backwards to a pre-translocated state. The rates and frequencies of these transitions vary significantly with DNA sequence and the magnitude of assisting force. During transcription pausing at an elemental pause sequence, we observe transitions between five distinct enzyme states (backtracked, pre-, half-, post-, and hyper-translocated), including a half-translocated state between pre and post. We develop a model for RNAP pausing and elongation by varying the applied force and monitoring RNAP mutants with SPRNT. Bowman 1 ; Richard H Ebright 3 ; Jens H Gundlach 1 ; 1 University of Washington, Physics, Seattle, WA, USA

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