Single-Molecule Studies of Repressor-DNA Interactions Show Long-Range Interactions

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...

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Published inProceedings of the National Academy of Sciences - PNAS Vol. 102; no. 28; pp. 9796 - 9801
Main Authors Wang, Y. M., Tegenfeldt, Jonas O., Reisner, W., Riehn, R., Guan, Xiao-Juan, Guo, Ling, Golding, Ido, Cox, Edward C., Sturm, James, Austin, Robert H.
Format Journal Article
LanguageEnglish
Published United States National Academy of Sciences 12.07.2005
National Acad Sciences
Subjects
Bacteriophage lambda - genetics
Biological Sciences
Biophysical Phenomena
Biophysics
Bleaching
Carbocyanines
Condensed Matter Physics
Den kondenserade materiens fysik
Deoxyribonucleic acid
Dimers
DNA
DNA, Viral - metabolism
DNA-Binding Proteins - isolation & purification
Dyes
Electrophoresis - methods
Fluorescence
Fysik
Green Fluorescent Proteins - metabolism
Imaging
Molecules
Monomers
Nanotechnology - methods
Natural Sciences
Naturvetenskap
Operator Regions, Genetic - genetics
Photons
Physical Sciences
Pixels
Proteins
Repressor Proteins - metabolism
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Summary: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.
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To whom correspondence should be addressed. E-mail: rha@suiling.princeton.edu.
Abbreviations: IPMC, integrated photon molecular counting; TIRF, total internal reflection fluorescence; POP-6, performance-optimized linear polyacrylamide; TBE, Tris/borate/EDTA.
Contributed by Robert H. Austin, April 29, 2005
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.
ISSN:0027-8424
1091-6490
1091-6490
DOI:10.1073/pnas.0502917102