Direct observation of one-dimensional diffusion and transcription by Escherichia coli RNA polymerase.
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 microM 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.
Guthold
M
M
Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403, USA.
Zhu
X
X
Rivetti
C
C
Yang
G
G
Thomson
N H
NH
Kasas
S
S
Hansma
H G
HG
Smith
B
B
Hansma
P K
PK
Bustamante
C
C
D000327:Adsorption; D000538:Aluminum Silicates; D002021:Buffers; D002413:Cations, Divalent / Q000494:pharmacology; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004058:Diffusion / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D017276:Friction; D006493:Heparin / Q000494:pharmacology; D007700:Kinetics; D018625:Microscopy, Atomic Force; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D013698:Templates, Genetic; D013728:Terminator Regions, Genetic / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics*
D000538:Aluminum Silicates; D002021:Buffers; D002413:Cations, Divalent; D006493:Heparin; D004247:DNA; D012321:DNA-Directed RNA Polymerases; C011934:mica