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

Overview

Journal Science

Specialty Science

Date 2007 May 26

PMID 17525339

Citations 459

Authors

Johan Elf

Gene-Wei Li

X Sunney Xie

Affiliations

Soon will be listed here.

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 approximately 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.

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References

Matthews K, Nichols J . Lactose repressor protein: functional properties and structure. Prog Nucleic Acid Res Mol Biol. 1997; 58:127-64. DOI: 10.1016/s0079-6603(08)60035-5. View

Ozbudak E, Thattai M, Kurtser I, Grossman A, van Oudenaarden A . Regulation of noise in the expression of a single gene. Nat Genet. 2002; 31(1):69-73. DOI: 10.1038/ng869. View

Winter R, Berg O, von Hippel P . Diffusion-driven mechanisms of protein translocation on nucleic acids. 3. The Escherichia coli lac repressor--operator interaction: kinetic measurements and conclusions. Biochemistry. 1981; 20(24):6961-77. DOI: 10.1021/bi00527a030. View

Lewis M . The lac repressor. C R Biol. 2005; 328(6):521-48. DOI: 10.1016/j.crvi.2005.04.004. View

von Hippel P, Berg O . Facilitated target location in biological systems. J Biol Chem. 1989; 264(2):675-8. View

Lewis M, Chang G, Horton N, Kercher M, Pace H, Schumacher M . Crystal structure of the lactose operon repressor and its complexes with DNA and inducer. Science. 1996; 271(5253):1247-54. DOI: 10.1126/science.271.5253.1247. View

Jacob F, MONOD J . Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol. 1961; 3:318-56. DOI: 10.1016/s0022-2836(61)80072-7. View

Kepes A . Transcription and translation in the lactose operon of Escherichia coli studied by in vivo kinetics. Prog Biophys Mol Biol. 1969; 19(1):199-236. DOI: 10.1016/0079-6107(69)90006-6. View

Bates D, Kleckner N . Chromosome and replisome dynamics in E. coli: loss of sister cohesion triggers global chromosome movement and mediates chromosome segregation. Cell. 2005; 121(6):899-911. PMC: 2973560. DOI: 10.1016/j.cell.2005.04.013. View

10.

Le T, Harlepp S, Guet C, Dittmar K, Emonet T, Pan T . Real-time RNA profiling within a single bacterium. Proc Natl Acad Sci U S A. 2005; 102(26):9160-4. PMC: 1166617. DOI: 10.1073/pnas.0503311102. View

11.

Blainey P, van Oijen A, Banerjee A, Verdine G, Xie X . A base-excision DNA-repair protein finds intrahelical lesion bases by fast sliding in contact with DNA. Proc Natl Acad Sci U S A. 2006; 103(15):5752-7. PMC: 1458645. DOI: 10.1073/pnas.0509723103. View

12.

Elowitz M, Levine A, Siggia E, Swain P . Stochastic gene expression in a single cell. Science. 2002; 297(5584):1183-6. DOI: 10.1126/science.1070919. View

13.

Oehler S, Eismann E, Kramer H, Muller-Hill B . The three operators of the lac operon cooperate in repression. EMBO J. 1990; 9(4):973-9. PMC: 551766. DOI: 10.1002/j.1460-2075.1990.tb08199.x. View

14.

Revzin A, Butler A, OConner P, Noble D, von Hippel P . Nonspecific DNA binding of genome-regulating proteins as a biological control mechanism: measurement of DNA-bound Escherichia coli lac repressor in vivo. Proc Natl Acad Sci U S A. 1977; 74(10):4228-32. PMC: 431912. DOI: 10.1073/pnas.74.10.4228. View

15.

Golding I, Paulsson J, Zawilski S, Cox E . Real-time kinetics of gene activity in individual bacteria. Cell. 2005; 123(6):1025-36. DOI: 10.1016/j.cell.2005.09.031. View

16.

Fieck A, Wyborski D, Short J . Modifications of the E.coli Lac repressor for expression in eukaryotic cells: effects of nuclear signal sequences on protein activity and nuclear accumulation. Nucleic Acids Res. 1992; 20(7):1785-91. PMC: 312271. DOI: 10.1093/nar/20.7.1785. View

17.

Dunaway M, Olson J, Rosenberg J, Kallai O, DICKERSON R, Matthews K . Kinetic studies of inducer binding to lac repressor.operator complex. J Biol Chem. 1980; 255(21):10115-9. View

18.

Wang A, Revzin A, Butler A, von Hippel P . Binding of E.coli lac repressor to non-operator DNA. Nucleic Acids Res. 1977; 4(5):1579-93. PMC: 343775. DOI: 10.1093/nar/4.5.1579. View

19.

Ozbudak E, Thattai M, Lim H, Shraiman B, van Oudenaarden A . Multistability in the lactose utilization network of Escherichia coli. Nature. 2004; 427(6976):737-40. DOI: 10.1038/nature02298. View

20.

Golding I, Cox E . Physical nature of bacterial cytoplasm. Phys Rev Lett. 2006; 96(9):098102. DOI: 10.1103/PhysRevLett.96.098102. View