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Science 316 (5828): 1191-1194

Copyright © 2007 by the American Association for the Advancement of Science

Probing Transcription Factor Dynamics at the Single-Molecule Level in a Living Cell

Johan Elf,1* Gene-Wei Li,2* X. Sunney Xie1{dagger}

Abstract: Transcription factors regulate gene expression through their binding to DNA. In a living Escherichia coli cell, we directly observed specific binding of a lac repressor, labeled with a fluorescent protein, to a chromosomal lac operator. Using single-molecule detection techniques, we measured the kinetics of binding and dissociation of the repressor in response to metabolic signals. Furthermore, we characterized the nonspecific binding to DNA, one-dimensional (1D) diffusion along DNA segments, and 3D translocation among segments through cytoplasm at the single-molecule level. In searching for the operator, a lac repressor spends ~90% of time nonspecifically bound to and diffusing along DNA with a residence time of <5 milliseconds. The methods and findings can be generalized to other nucleic acid binding proteins.

1 Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
2 Department of Physics, Harvard University, Cambridge, MA 02138, USA.

* These authors contributed equally to this work.

{dagger} To whom correspondence should be addressed. E-mail: xie{at}

Gene location and DNA density determine transcription factor distributions in Escherichia coli.
T. E. Kuhlman and E. C. Cox (2014)
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   Abstract »    Full Text »    PDF »
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   Abstract »    Full Text »    PDF »
Physical constraints determine the logic of bacterial promoter architectures.
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   Abstract »    Full Text »    PDF »
Androgen receptor complexes probe DNA for recognition sequences by short random interactions.
M. E. Van Royen, W. A. van Cappellen, B. Geverts, T. Schmidt, A. B. Houtsmuller, and M. J. M. Schaaf (2014)
J. Cell Sci. 127, 1406-1416
   Abstract »    Full Text »    PDF »
Impacts of the ubiquitous factor Zelda on Bicoid-dependent DNA binding and transcription in Drosophila.
Z. Xu, H. Chen, J. Ling, D. Yu, P. Struffi, and S. Small (2014)
Genes & Dev. 28, 608-621
   Abstract »    Full Text »    PDF »
Quantitative influence of macromolecular crowding on gene regulation kinetics.
M. Tabaka, T. Kalwarczyk, and R. Holyst (2014)
Nucleic Acids Res. 42, 727-738
   Abstract »    Full Text »    PDF »
Transcription-factor binding and sliding on DNA studied using micro- and macroscopic models.
E. G. Marklund, A. Mahmutovic, O. G. Berg, P. Hammar, D. van der Spoel, D. Fange, and J. Elf (2013)
PNAS 110, 19796-19801
   Abstract »    Full Text »    PDF »
Building synthetic cellular organization.
J. K. Polka and P. A. Silver (2013)
Mol. Biol. Cell 24, 3585-3587
   Abstract »    Full Text »    PDF »
Measuring Chromatin Interaction Dynamics on the Second Time Scale at Single-Copy Genes.
K. Poorey, R. Viswanathan, M. N. Carver, T. S. Karpova, S. M. Cirimotich, J. G. McNally, S. Bekiranov, and D. T. Auble (2013)
Science 342, 369-372
   Abstract »    Full Text »    PDF »
Molecular crowding limits translation and cell growth.
S. Klumpp, M. Scott, S. Pedersen, and T. Hwa (2013)
PNAS 110, 16754-16759
   Abstract »    Full Text »    PDF »
The autoregulation of a eukaryotic DNA transposon.
C. Claeys Bouuaert, K. Lipkow, S. S. Andrews, D. Liu, and R. Chalmers (2013)
eLife Sci 2, e00668
   Abstract »    Full Text »    PDF »
Single-molecule DNA repair in live bacteria.
S. Uphoff, R. Reyes-Lamothe, F. Garza de Leon, D. J. Sherratt, and A. N. Kapanidis (2013)
PNAS 110, 8063-8068
   Abstract »    Full Text »    PDF »
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S. Kim, E. Brostromer, D. Xing, J. Jin, S. Chong, H. Ge, S. Wang, C. Gu, L. Yang, Y. Q. Gao, et al. (2013)
Science 339, 816-819
   Abstract »    Full Text »    PDF »
V. Graziano, G. Luo, P. C. Blainey, A. J. Perez-Berna, W. J. McGrath, S. J. Flint, C. San Martin, X. S. Xie, and W. F. Mangel (2013)
J. Biol. Chem. 288, 2068-2080
   Abstract »    Full Text »    PDF »
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P. C. Blainey, V. Graziano, A. J. Perez-Berna, W. J. McGrath, S. J. Flint, C. San Martin, X. S. Xie, and W. F. Mangel (2013)
J. Biol. Chem. 288, 2092-2102
   Abstract »    Full Text »    PDF »
DNA regions bound at low occupancy by transcription factors do not drive patterned reporter gene expression in Drosophila.
W. W. Fisher, J. J. Li, A. S. Hammonds, J. B. Brown, B. D. Pfeiffer, R. Weiszmann, S. MacArthur, S. Thomas, J. A. Stamatoyannopoulos, M. B. Eisen, et al. (2012)
PNAS 109, 21330-21335
   Abstract »    Full Text »    PDF »
High-throughput gene expression analysis at the level of single proteins using a microfluidic turbidostat and automated cell tracking.
G. Ullman, M. Wallden, E. G. Marklund, A. Mahmutovic, I. Razinkov, and J. Elf (2012)
Phil Trans R Soc B 368, 20120025
   Abstract »    Full Text »    PDF »
The physics of life: one molecule at a time.
M. C. Leake (2012)
Phil Trans R Soc B 368, 20120248
   Abstract »    Full Text »    PDF »
Biologistics--Diffusion coefficients for complete proteome of Escherichia coli.
T. Kalwarczyk, M. Tabaka, and R. Holyst (2012)
Bioinformatics 28, 2971-2978
   Abstract »    Full Text »    PDF »
Sequence-dependent sliding kinetics of p53.
J. S. Leith, A. Tafvizi, F. Huang, W. E. Uspal, P. S. Doyle, A. R. Fersht, L. A. Mirny, and A. M. van Oijen (2012)
PNAS 109, 16552-16557
   Abstract »    Full Text »    PDF »
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A. Bancaud, C. Lavelle, S. Huet, and J. Ellenberg (2012)
Nucleic Acids Res. 40, 8783-8792
   Abstract »    Full Text »    PDF »
The magnitude and colour of noise in genetic negative feedback systems.
M. Voliotis and C. G. Bowsher (2012)
Nucleic Acids Res. 40, 7084-7095
   Abstract »    Full Text »    PDF »
Conditional cooperativity in toxin-antitoxin regulation prevents random toxin activation and promotes fast translational recovery.
I. Cataudella, A. Trusina, K. Sneppen, K. Gerdes, and N. Mitarai (2012)
Nucleic Acids Res. 40, 6424-6434
   Abstract »    Full Text »    PDF »
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D. Mazza, A. Abernathy, N. Golob, T. Morisaki, and J. G. McNally (2012)
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   Abstract »    Full Text »    PDF »
Galactose repressor mediated intersegmental chromosomal connections in Escherichia coli.
Z. Qian, E. K. Dimitriadis, R. Edgar, P. Eswaramoorthy, and S. Adhya (2012)
PNAS 109, 11336-11341
   Abstract »    Full Text »    PDF »
The lac Repressor Displays Facilitated Diffusion in Living Cells.
P. Hammar, P. Leroy, A. Mahmutovic, E. G. Marklund, O. G. Berg, and J. Elf (2012)
Science 336, 1595-1598
   Abstract »    Full Text »    PDF »
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A. Benabbas, V. Karunakaran, H. Youn, T. L. Poulos, and P. M. Champion (2012)
J. Biol. Chem. 287, 21729-21740
   Abstract »    Full Text »    PDF »
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T. Lenn and M. C. Leake (2012)
Open Bio 2, 120090
   Abstract »    Full Text »    PDF »
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N. R. Zabet and B. Adryan (2012)
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   Abstract »    Full Text »    PDF »
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N. R. Zabet and B. Adryan (2012)
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   Abstract »    Full Text »    PDF »
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A. Millius, N. Watanabe, and O. D. Weiner (2012)
J. Cell Sci. 125, 1165-1176
   Abstract »    Full Text »    PDF »
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G. Lia, B. Michel, and J.-F. Allemand (2012)
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   Abstract »    Full Text »    PDF »
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M. L. Ferguson, D. Le Coq, M. Jules, S. Aymerich, O. Radulescu, N. Declerck, and C. A. Royer (2012)
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   Abstract »    Full Text »    PDF »
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Z. Burda, A. Krzywicki, O. C. Martin, and M. Zagorski (2011)
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   Abstract »    Full Text »    PDF »
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A. Sanchez, M. L. Osborne, L. J. Friedman, J. Kondev, and J. Gelles (2011)
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   Abstract »    Full Text »    PDF »
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L. Wang, J. S. Brugge, and K. A. Janes (2011)
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   Abstract »    Full Text »    PDF »
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B. P. Bratton, R. A. Mooney, and J. C. Weisshaar (2011)
J. Bacteriol. 193, 5138-5146
   Abstract »    Full Text »    PDF »
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W. Wang, G.-W. Li, C. Chen, X. S. Xie, and X. Zhuang (2011)
Science 333, 1445-1449
   Abstract »    Full Text »    PDF »
Single-molecule investigations of the stringent response machinery in living bacterial cells.
B. P. English, V. Hauryliuk, A. Sanamrad, S. Tankov, N. H. Dekker, and J. Elf (2011)
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   Abstract »    Full Text »    PDF »
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T. J. Petty, S. Emamzadah, L. Costantino, I. Petkova, E. S. Stavridi, J. G. Saven, E. Vauthey, and T. D. Halazonetis (2011)
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   Abstract »    Full Text »    PDF »
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   Abstract »    Full Text »    PDF »
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   Abstract »    PDF »
Concentration-dependent exchange accelerates turnover of proteins bound to double-stranded DNA.
J. S. Graham, R. C. Johnson, and J. F. Marko (2011)
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   Abstract »    Full Text »    PDF »
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S. A. Isaacson, D. M. McQueen, and C. S. Peskin (2011)
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   Abstract »    Full Text »    PDF »
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A. Tafvizi, F. Huang, A. R. Fersht, L. A. Mirny, and A. M. van Oijen (2011)
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   Abstract »    Full Text »    PDF »
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A. D. Jeyasekharan, N. Ayoub, R. Mahen, J. Ries, A. Esposito, E. Rajendra, H. Hattori, R. P. Kulkarni, and A. R. Venkitaraman (2010)
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   Abstract »    Full Text »    PDF »
Classic and contemporary approaches to modeling biochemical reactions.
W. W. Chen, M. Niepel, and P. K. Sorger (2010)
Genes & Dev. 24, 1861-1875
   Abstract »    Full Text »    PDF »
Imaging OmpR Binding to Native Chromosomal Loci in Escherichia coli.
E. A. Libby, S. Ekici, and M. Goulian (2010)
J. Bacteriol. 192, 4045-4053
   Abstract »    Full Text »    PDF »
D. Phillips, M. J. Reilley, A. M. Aponte, G. Wang, E. Boja, M. Gucek, and R. S. Balaban (2010)
J. Biol. Chem. 285, 23532-23536
   Abstract »    Full Text »    PDF »
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T. G. W. Graham, S. M. A. Tabei, A. R. Dinner, and I. Rebay (2010)
Development 137, 2265-2278
   Abstract »    Full Text »    PDF »
Experimental snapshots of a protein-DNA binding landscape.
I. E. Sanchez, D. U. Ferreiro, M. Dellarole, and G. de Prat-Gay (2010)
PNAS 107, 7751-7756
   Abstract »    Full Text »    PDF »
Site-specific chromosomal integration of large synthetic constructs.
T. E. Kuhlman and E. C. Cox (2010)
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   Abstract »    Full Text »    PDF »
Quantitative study of synthetic Hox transcription factor-DNA interactions in live cells.
V. Vukojevic, D. K. Papadopoulos, L. Terenius, W. J. Gehring, and R. Rigler (2010)
PNAS 107, 4093-4098
   Abstract »    Full Text »    PDF »
Spatio-temporal correlations can drastically change the response of a MAPK pathway.
K. Takahashi, S. Tanase-Nicola, and P. R. ten Wolde (2010)
PNAS 107, 2473-2478
   Abstract »    Full Text »    PDF »
Actin polymerization driven mitochondrial transport in mating S. cerevisiae.
E. N. Senning and A. H. Marcus (2010)
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   Abstract »    Full Text »    PDF »
Transcriptional regulation shapes the organization of genes on bacterial chromosomes.
S. C. Janga, H. Salgado, and A. Martinez-Antonio (2009)
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   Abstract »    Full Text »    PDF »
Facilitated diffusion with DNA coiling.
M. A. Lomholt, B. van den Broek, S.-M. J. Kalisch, G. J. L. Wuite, and R. Metzler (2009)
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   Abstract »    Full Text »    PDF »
Assembly of multiprotein complexes that control genome function.
C. Dinant, M. S. Luijsterburg, T. Hofer, G. von Bornstaedt, W. Vermeulen, A. B. Houtsmuller, and R. van Driel (2009)
J. Cell Biol. 185, 21-26
   Abstract »    Full Text »    PDF »
MetJ repressor interactions with DNA probed by in-cell NMR.
A. M. Augustus, P. N. Reardon, and L. D. Spicer (2009)
PNAS 106, 5065-5069
   Abstract »    Full Text »    PDF »
Bioinformatics Resources for the Study of Gene Regulation in Bacteria.
J. Collado-Vides, H. Salgado, E. Morett, S. Gama-Castro, V. Jimenez-Jacinto, I. Martinez-Flores, A. Medina-Rivera, L. Muniz-Rascado, M. Peralta-Gil, and A. Santos-Zavaleta (2009)
J. Bacteriol. 191, 23-31
   Full Text »    PDF »
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M. C. Konopka, K. A. Sochacki, B. P. Bratton, I. A. Shkel, M. T. Record, and J. C. Weisshaar (2009)
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   Abstract »    Full Text »    PDF »
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S. Klumpp and T. Hwa (2008)
PNAS 105, 20245-20250
   Abstract »    Full Text »    PDF »
Extinction and resurrection in gene networks.
D. Schultz, A. M. Walczak, J. N. Onuchic, and P. G. Wolynes (2008)
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   Abstract »    Full Text »    PDF »
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Science 322, 1092-1097
   Abstract »    Full Text »    PDF »
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P. J. Choi, L. Cai, K. Frieda, and X. S. Xie (2008)
Science 322, 442-446
   Abstract »    Full Text »    PDF »
Global jumping and domain-specific intersegment transfer between DNA cognate sites of the multidomain transcription factor Oct-1.
M. Doucleff and G. M. Clore (2008)
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   Abstract »    Full Text »    PDF »
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I. Bonnet, A. Biebricher, P.-L. Porte, C. Loverdo, O. Benichou, R. Voituriez, C. Escude, W. Wende, A. Pingoud, and P. Desbiolles (2008)
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   Abstract »    Full Text »    PDF »
Spatial effects on the speed and reliability of protein-DNA search.
Z. Wunderlich and L. A. Mirny (2008)
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   Abstract »    Full Text »    PDF »
How gene order is influenced by the biophysics of transcription regulation.
G. Kolesov, Z. Wunderlich, O. N. Laikova, M. S. Gelfand, and L. A. Mirny (2007)
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   Abstract »    Full Text »    PDF »
Label-Free, Single-Molecule Detection with Optical Microcavities.
A. M. Armani, R. P. Kulkarni, S. E. Fraser, R. C. Flagan, and K. J. Vahala (2007)
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   Abstract »    Full Text »    PDF »
Imaging individual transcription factors.
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   Full Text »    PDF »

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