Quantitative transcription factor binding kinetics at the single-molecule level.
We investigated the binding interaction between the bacteriophage lambda-repressor CI and its target DNA using total internal reflection fluorescence microscopy. Large stepwise changes in the intensity of the red fluorescent protein fused to CI were observed as it associated with and dissociated from individually labeled single-molecule DNA targets. The stochastic association and dissociation were characterized by Poisson statistics. Dark and bright intervals were measured for thousands of individual events. The exponential distribution of the intervals allowed direct determination of the association and dissociation rate constants (k(a) and k(d), respectively). We resolved in detail how k(a) and k(d) varied as a function of three control parameters: the DNA length L, the CI dimer concentration, and the binding affinity. Our results show that although interactions with nonoperator DNA sequences are observable, CI binding to the operator site is not dependent on the length of flanking nonoperator DNA.
Wang
Yufang
Y
Department of Physics, Princeton University, Princeton, New Jersey, USA.
Guo
Ling
L
Golding
Ido
I
Cox
Edward C
EC
Ong
N P
NP
D010582:Bacteriophage lambda; D004247:DNA / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins; D007091:Image Processing, Computer-Assisted; D007700:Kinetics; D008164:Luminescent Proteins; D008856:Microscopy, Fluorescence; D015233:Models, Statistical; D009875:Operator Regions, Genetic; D016012:Poisson Distribution; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism*; D013269:Stochastic Processes; D014157:Transcription Factors / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins / Q000378:metabolism*; D000097573:Red Fluorescent Protein
D004247:DNA; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D054334:Viral Regulatory and Accessory Proteins; D000097573:Red Fluorescent Protein; C016649:phage repressor proteins