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Biophys J, October 1999, p. 2284-2294, Vol. 77, No. 4
*Institute of Molecular Biology, #Physics Department,
§Howard Hughes Medical Institute, and
Chemistry Department, University of Oregon, Eugene,
Oregon 97403; and ¶Physics Department, University of
California, Santa Barbara, California 93106 USA
The dynamics of nonspecific and specific
Escherichia coli RNA polymerase (RNAP)-DNA complexes
have been directly observed using scanning force microscopy operating
in buffer. To this end, imaging conditions had to be found in which DNA
molecules were adsorbed onto mica strongly enough to be imaged, but
loosely enough to be able to diffuse on the surface. In sequential
images of nonspecific complexes, RNAP was seen to slide along DNA,
performing a one-dimensional random walk. Heparin, a substance known to
disrupt nonspecific RNAP-DNA interactions, prevented sliding. These
observations suggest that diffusion of RNAP along DNA constitutes a
mechanism for accelerated promoter location. Sequential images of
single, transcribing RNAP molecules were also investigated. Upon
addition of 5 µM nucleoside triphosphates to stalled
elongation complexes in the liquid chamber, RNAP molecules were seen to
processively thread their template at rates of 1.5 nucleotide/s in a
direction consistent with the promoter orientation. Transcription
assays, performed with radiolabeled, mica-bound transcription
complexes, confirmed this rate, which was about three times smaller
than the rate of complexes in solution. This assay also showed that the
pattern of pause sites and the termination site were affected by the
surface. By using the Einstein-Sutherland friction-diffusion relation
the loading force experienced by RNAP due to DNA-surface friction is
estimated and discussed.
Biophys J, October 1999, p. 2284-2294, Vol. 77, No. 4
© 1999 by the Biophysical Society 0006-3495/99/10/2284/11 $2.00
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