Mr. William J. Greenleaf Profile

William J. Greenleaf

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Proceedings Article | 5 September 2007 Paper

High-resolution single-molecule optical trapping measurements of transcription with basepair accuracy: instrumentation and methods

William Greenleaf, Kirsten Frieda, Elio Abbondanzieri, Michael Woodside, Steven Block

Proceedings Volume 6644, 664406 (2007) https://doi.org/10.1117/12.739631

KEYWORDS: Optical tweezers, Polymers, Calibration, Molecules, Optical testing, Motion measurement, Sensors, Helium, Dielectrics, Acousto-optics

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Optical traps allow the single-molecule investigation of the chemo-mechanical properties of biomolecules¹. We have developed an ultra-stable optical trapping system capable of ångstr&diaero;m-level position resolution and used it to monitor transcriptional elongation by single molecules of E. coli RNAP polymerase (RNAP). This optical trapping system uses the anharmonic region of the trapping potential, where differential stiffness vanishes, to generate a force-clamp that operates without feedback-associated noise². We demonstrate methods of calibrating this anharmonic trapping region and strategies to eliminate common sources of noise associated with air currents. Records of transcriptional elongation obtained with this device showed discrete steps averaging ~3.7 Å, a distance equivalent to the mean rise per base found in B-DNA³. To determine the absolute position of the RNAP on the DNA template, we monitored transcription under conditions in which a single nucleotide species was held rate-limiting and then aligned the resulting transcriptional pauses with the occurrence of this rate-limiting species in the underlying template. The aligned pause patterns recorded from four molecules, each measured with a different ratelimiting nucleotide species, were used to determine the sequence of a short region of unknown DNA, demonstrating that the motion of a single processive nucleic acid enzyme may be used to extract sequence information directly from DNA⁴.

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