Escherichia coli RNA polymerase activity observed using atomic force microscopy.
Fluid tapping-mode atomic force microscopy (AFM) was used to observe Escherichia coli RNA polymerase (RNAP) transcribing two different linear double-stranded (ds) DNA templates. The transcription process was detected by observing the translocation of the DNA template by RNAP on addition of ribonucleoside 5'-triphosphates (NTPs) in sequential AFM images. Stalled ternary complexes of RNAP, dsDNA and nascent RNA were adsorbed onto a mica surface and imaged under continuously flowing buffer. On introduction of all four NTPs, we observed some DNA molecules being pulled through the RNAP, some dissociating from the RNAP and others which did not move relative to the RNAP. The transcription rates were observed to be approximately 0.5-2 bases/s at our NTP concentrations, approximately 5 microM. The RNA transcripts were not unambiguously imaged in fluid. However, in experiments using a small single-stranded (ss) circular DNA template, known as a rolling circle, transcripts up to 1 or 2 microns long could be observed with tapping mode AFM once the samples were dried and imaged in air. This confirmed our observations of the transcriptional activity of RNAP adsorbed onto mica. This work illustrates that the development of tapping-mode in fluid has made it possible to use AFM to follow biological processes at the molecular level and get new insights about the variability of activity of individual molecules bound to a surface.
Kasas
S
S
Physics Department, University of California Santa Barbara 93106, USA.
Thomson
N H
NH
Smith
B L
BL
Hansma
H G
HG
Zhu
X
X
Guthold
M
M
Bustamante
C
C
Kool
E T
ET
Kashlev
M
M
Hansma
P K
PK
D000538:Aluminum Silicates; D002021:Buffers; D004247:DNA / Q000378:metabolism; D004270:DNA, Circular / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D018625:Microscopy, Atomic Force; D013698:Templates, Genetic; D014158:Transcription, Genetic; D015032:Zinc
D000538:Aluminum Silicates; D002021:Buffers; D004270:DNA, Circular; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D015032:Zinc; C011934:mica