Single-molecule studies of repressor–DNA interactions show long-range interactions

doi:10.1073/pnas.0502917102

Single-molecule studies of repressor–DNA interactions show long-range interactions

Departments of ^*Physics, ^†Molecular Biology, and ^‡Electrical Engineering, Princeton University, Princeton, NJ 08544

Contributed by Robert H. Austin, April 29, 2005

Abstract

We have performed single-molecule studies of GFP–LacI repressor proteins bound to bacteriophage λ DNA containing a 256 tandem lac operator insertion confined in nanochannels. An integrated photon molecular counting method was developed to determine the number of proteins bound to DNA. By using this method, we determined the saturated mean occupancy of the 256 tandem lac operators to be 13, which constitutes only 2.5% of the available sites. This low occupancy level suggests that the repressors influence each other even when they are widely separated, at distances on the order of 200 nm, or several DNA persistence lengths.

Footnotes

↵ § To whom correspondence should be addressed. E-mail: rha{at}suiling.princeton.edu.
Author contributions: Y.M.W., J.O.T., W.R., R.R., E.C.C., J.S., and R.H.A. designed research; Y.M.W., W.R., E.C.C., and R.H.A. analyzed data; W.R., R.R., X.-J.G., L.G., I.G., E.C.C., and J.S. contributed new reagents/analytic tools; and Y.M.W., J.O.T., W.R., E.C.C., and R.H.A. wrote the paper.
Abbreviations: IPMC, integrated photon molecular counting; TIRF, total internal reflection fluorescence; POP-6, performance-optimized linear polyacrylamide; TBE, Tris/borate/EDTA.
Freely available online through the PNAS open access option.