Direct observation of one-dimensional diffusion and transcription by Escherichia coli RNA polymerase

The dynamics of nonspecific and specific Escherichia coli RNA polymerase (R NAP)-DNA complexes have been directly observed using scanning force microsc opy 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 an the surface. In sequential imag es of nonspecific complexes, RNAP was seen to slide along DNA, performing a one-dimensional random walk. Heparin, a substance known to disrupt nonspec ific RNAP-DNA interactions, prevented sliding. These observations suggest t hat diffusion of RNAP along DNA constitutes a mechanism for accelerated pro moter location. Sequential images of single, transcribing RNAP molecules we re also investigated. Upon addition of 5 mu M nucleoside triphosphates to s talled elongation complexes in the liquid chamber, RNAP molecules were seen to processively thread their template at rates of 1.5 nucleotide/s in a di rection consistent with the promoter orientation. Transcription assays, per formed with radiolabeled, mica-bound transcription complexes, confirmed thi s rate, which was about three times smaller than the rate of complexes in s olution. This assay also showed that the pattern of pause sites and the ter mination site were affected by the surface. By using the Einstein-Sutherlan d friction-diffusion relation the loading force experienced by RNAP due to DNA-surface friction is estimated and discussed.