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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