Quantitative transcription factor binding kinetics at the single-molecule level

Yufang Wang, Ling Guo, Ido Golding, Edward C. Cox, N. P. Ong

Research output: Contribution to journalArticle

45 Scopus citations

Abstract

We investigated the binding interaction between the bacteriophage l-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 (ka and kd, respectively). We resolved in detail how ka and kd 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.

Original languageEnglish (US)
Pages (from-to)609-620
Number of pages12
JournalBiophysical Journal
Volume96
Issue number2
DOIs
StatePublished - Jan 21 2009

All Science Journal Classification (ASJC) codes

  • Biophysics

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