Following the assembly of RNA polymerase-DNA complexes in aqueous solutions with the scanning force microscope.
The capability of the scanning force microscope (SFM) to image molecules in aqueous buffers has opened the exciting possibility of following processes of molecular assembly in real time and in near-physiological environments. This capability is demonstrated in this paper by following the assembly process of RNA polymerase-DNA complexes. DNA fragments deposited on mica and imaged in Hepes/MgCl2 are shown before and after Escherichia coli RNA polymerase holoenzyme is injected in the SFM liquid chamber. The protein can recognize and bind to these DNA fragments within several seconds after injection, suggesting that the protein and the DNA retain their native configuration after deposition and during SFM imaging. A time-lapse sequence depicting the process of assembly of RNA polymerase-DNA complexes is shown. These results represent the first step for acquiring the capabilities to monitor complex biomolecular processes as they take place in ionic solutions and to characterize their spatial organization.
Guthold
M
M
Institute of Molecular Biology, University of Oregon, Eugene 97403.
Bezanilla
M
M
Erie
D A
DA
Jenkins
B
B
Hansma
H G
HG
Bustamante
C
C
D001426:Bacterial Proteins; D010582:Bacteriophage lambda; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D066298:In Vitro Techniques; D018625:Microscopy, Atomic Force / Q000379:methods*; D011401:Promoter Regions, Genetic; D012996:Solutions; D013997:Time Factors
D001426:Bacterial Proteins; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D012996:Solutions; D012321:DNA-Directed RNA Polymerases