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Proceedings Paper

High-resolution single-molecule optical trapping measurements of transcription with basepair accuracy: instrumentation and methods
Author(s): William J. Greenleaf; Kirsten L Frieda; Elio A. Abbondanzieri; Michael T. Woodside; Steven M. Block
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Paper Abstract

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.

Paper Details

Date Published: 5 September 2007
PDF: 8 pages
Proc. SPIE 6644, Optical Trapping and Optical Micromanipulation IV, 664406 (5 September 2007); doi: 10.1117/12.739631
Show Author Affiliations
William J. Greenleaf, Stanford Univ. (United States)
Kirsten L Frieda, Stanford Univ. (United States)
Elio A. Abbondanzieri, Stanford Univ. (United States)
Michael T. Woodside, Univ. of Alberta (Canada)
National Research Council of Canada (Canada)
Steven M. Block, Stanford Univ. (United States)

Published in SPIE Proceedings Vol. 6644:
Optical Trapping and Optical Micromanipulation IV
Kishan Dholakia; Gabriel C. Spalding, Editor(s)

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