Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 115, Issue 6, Pages 1286-1291Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1719906115
Keywords
optical tweezers; transcription; single molecule; step-finding
Categories
Funding
- Howard Hughes Medical Institute
- NIH [R01GM032543, R01GM071552]
- US Department of Energy Office of Basic Energy Sciences Nanomachine Program [DE-AC02-05CH11231]
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In recent years, highly stable optical tweezers systems have enabled the characterization of the dynamics of molecular motors at very high resolution. However, the motion of many motors with angstrom-scale dynamics cannot be consistently resolved due to poor signal-to-noise ratio. Using an acousto-optic deflector to generate a time-shared dual-optical trap, we decreased low-frequency noise by more than one order of magnitude compared with conventional dual-trap optical tweezers. Using this instrument, we implemented a protocol that synthesizes single base-pair trajectories, which are used to test a Large State Space Hidden Markov Model algorithm to recover their individual steps. We then used this algorithm on real transcription data obtained in the same instrument to fully uncover the molecular trajectories of Escherichia coli RNA polymerase. We applied this procedure to reveal the effect of pyrophosphate on the distribution of dwell times between consecutive polymerase steps.
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