Abstract
Gene expression originates from individual DNA molecules within living cells. Like many single-molecule processes, gene expression and regulation are stochastic, that is, sporadic in time. This leads to heterogeneity in the messenger-RNA and protein copy numbers in a population of cells with identical genomes. With advanced single-cell fluorescence microscopy, it is now possible to quantify transcriptomes and proteomes with single-molecule sensitivity. Dynamic processes such as transcription-factor binding, transcription and translation can be monitored in real time, providing quantitative descriptions of the central dogma of molecular biology and the demonstration that a stochastic single-molecule event can determine the phenotype of a cell.
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References
Hirschfeld, T. Optical microscopic observation of single small molecules. Appl. Opt. 15, 2965–2966 (1976). This paper presents the first single-molecule fluorescence imaging at room temperature, and the approach, which reduces the detection volume to enhance signal-to-background ratio, is widely used today in vitro and in living cells.
Shera, E. B., Seitzinger, N. K., Davis, L. M., Keller, R. A. & Soper, S. A. Detection of single fluorescent molecules. Chem. Phys. Lett. 174, 553–557 (1990).
Betzig, E. & Chichester, R. J. Single molecules observed by near-field scanning optical microscopy. Science 262, 1422–1425 (1993).
Funatsu, T., Harada, Y., Tokunaga, M., Saito, K. & Yanagida, T. Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution. Nature 374, 555–559 (1995).
Schmidt, T., Schutz, G. J., Baumgartner, W., Gruber, H. J. & Schindler, H. Characterization of photophysics and mobility of single molecules in a fluid lipid-membrane. J. Phys. Chem. 99, 17662–17668 (1995).
Xie, X. S. & Trautman, J. K. Optical studies of single molecules at room temperature. Annu. Rev. Phys. Chem. 49, 441–480 (1998).
Moerner, W. E. & Orrit, M. Illuminating single molecules in condensed matter. Science 283, 1670–1676 (1999).
Ha, T. et al. Single-molecule fluorescence spectroscopy of enzyme conformational dynamics and cleavage mechanism. Proc. Natl Acad. Sci. USA 96, 893–898 (1999).
Lu, H. P., Xun, L. & Xie, X. S. Single-molecule enzymatic dynamics. Science 282, 1877–1882 (1998). This article reports real-time observation and statistical analyses of stochastic enzymatic turnover and shows that on a single-molecule basis, k cat is not a constant.
Hamill, O. P., Marty, A., Neher, E., Sakmann, B. & Sigworth, F. J. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch. 391, 85–100 (1981).
Eid, J. et al. Real-time DNA sequencing from single polymerase molecules. Science 323, 133–138 (2009). This paper reports real-time sequencing of DNA using a single DNA polymerase molecule, a compelling application of single-molecule enzymology in biotechnology.
Bouche, J. P. Physical map of a 470 × 103 base-pair region flanking the terminus of DNA replication in the Escherichia coli K12 genome. J. Mol. Biol. 154, 1–20 (1982).
Bremer, H. A stochastic process determines the time at which cell division begins in Escherichia coli . J. Theor. Biol. 118, 351–365 (1986).
Wang, M. D. et al. Force and velocity measured for single molecules of RNA polymerase. Science 282, 902–907 (1998).
Blanchard, S. C., Kim, H. D., Gonzalez, R. L. Jr, Puglisi, J. D. & Chu, S. tRNA dynamics on the ribosome during translation. Proc. Natl Acad. Sci. USA 101, 12893–12898 (2004).
Kapanidis, A. N. et al. Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism. Science 314, 1144–1147 (2006).
Wen, J. D. et al. Following translation by single ribosomes one codon at a time. Nature 452, 598–603 (2008).
Cornish, P. V., Ermolenko, D. N., Noller, H. F. & Ha, T. Spontaneous intersubunit rotation in single ribosomes. Mol. Cell 30, 578–588 (2008).
Bernstein, J. A., Khodursky, A. B., Lin, P. H., Lin-Chao, S. & Cohen, S. N. Global analysis of mRNA decay and abundance in Escherichia coli at single-gene resolution using two-color fluorescent DNA microarrays. Proc. Natl Acad. Sci. USA 99, 9697–9702 (2002).
Ishihama, Y. et al. Protein abundance profiling of the Escherichia coli cytosol. BMC Genomics 9, 102 (2008).
Taniguchi, Y. et al. Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells. Science 329, 533–538 (2010). This article profiles the transcriptome and proteome with single-molecule sensitivity in single cells, and shows that the protein copy-number distribution of most genes can be well fit with gamma distribution.
Xie, X. S., Choi, P. J., Li, G. W., Lee, N. K. & Lia, G. Single-molecule approach to molecular biology in living bacterial cells. Annu. Rev. Biophys. 37, 417–444 (2008).
Giepmans, B. N., Adams, S. R., Ellisman, M. H. & Tsien, R. Y. The fluorescent toolbox for assessing protein location and function. Science 312, 217–224 (2006).
Andersson, H., Baechi, T., Hoechl, M. & Richter, C. Autofluorescence of living cells. J. Microsc. 191, 1–7 (1998).
Sako, Y., Minoghchi, S. & Yanagida, T. Single-molecule imaging of EGFR signalling on the surface of living cells. Nature Cell Biol. 2, 168–172 (2000).
Iino, R., Koyama, I. & Kusumi, A. Single molecule imaging of green fluorescent proteins in living cells: E-cadherin forms oligomers on the free cell surface. Biophys. J. 80, 2667–2677 (2001).
Wilson, T. Confocal Microscopy (Academic, 1990).
Denk, W., Strickler, J. H. & Webb, W. W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).
Sanchez, E. J., Novotny, L., Holtom, G. R. & Xie, X. S. Room-temperature fluorescence imaging and spectroscopy of single molecules by two-photon excitation. J. Phys. Chem. A 101, 7019–7023 (1997).
Fuchs, E., Jaffe, J., Long, R. & Azam, F. Thin laser light sheet microscope for microbial oceanography. Opt. Express 10, 145–154 (2002).
Ritter, J. G., Veith, R., Veenendaal, A., Siebrasse, J. P. & Kubitscheck, U. Light sheet microscopy for single molecule tracking in living tissue. PLoS ONE 5, e11639 (2010).
Elowitz, M. B., Surette, M. G., Wolf, P. E., Stock, J. B. & Leibler, S. Protein mobility in the cytoplasm of Escherichia coli . J. Bacteriol. 181, 197–203 (1999).
Xie, X. S., Yu, J. & Yang, W. Y. Living cells as test tubes. Science 312, 228–230 (2006).
Yu, J., Xiao, J., Ren, X., Lao, K. & Xie, X. S. Probing gene expression in live cells, one protein molecule at a time. Science 311, 1600–1603 (2006). This article monitors the real-time production of individual proteins by imaging newly synthesized YFP molecules, demonstrating translational bursts from a repressed promoter.
Elf, J., Li, G. W. & Xie, X. S. Probing transcription factor dynamics at the single-molecule level in a living cell. Science 316, 1191–1194 (2007). This article probes specific and nonspecific binding of a transcription factor to DNA, and measures the search time for a transcription factor to reach its target site in a living bacterial cell.
Thompson, R. E., Larson, D. R. & Webb, W. W. Precise nanometer localization analysis for individual fluorescent probes. Biophys. J. 82, 2775–2783 (2002).
Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nature Methods 3, 793–795 (2006).
Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).
Hess, S. T., Girirajan, T. P. & Mason, M. D. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys. J. 91, 4258–4272 (2006).
Hell, S. W. Far-field optical nanoscopy. Science 316, 1153–1158 (2007).
Richter, P. H. & Eigen, M. Diffusion controlled reaction rates in spheroidal geometry: application to repressor–operator association and membrane bound enzymes. Biophys. Chem. 2, 255–263 (1974).
Berg, O. G., Winter, R. B. & von Hippel, P. H. Diffusion-driven mechanisms of protein translocation on nucleic acids. 1. Models and theory. Biochemistry 20, 6929–6948 (1981).
Riggs, A. D., Bourgeois, S. & Cohn, M. The lac repressor–operator interaction. 3. Kinetic studies. J. Mol. Biol. 53, 401–417 (1970).
Kabata, H. et al. Visualization of single molecules of RNA polymerase sliding along DNA. Science 262, 1561–1563 (1993).
Harada, Y. et al. Single-molecule imaging of RNA polymerase-DNA interactions in real time. Biophys. J. 76, 709–715 (1999).
Blainey, P. C., van Oijen, A. M., Banerjee, A., Verdine, G. L. & Xie, X. S. A base-excision DNA-repair protein finds intrahelical lesion bases by fast sliding in contact with DNA. Proc. Natl Acad. Sci. USA 103, 5752–5757 (2006).
Graneli, A., Yeykal, C. C., Robertson, R. B. & Greene, E. C. Long-distance lateral diffusion of human Rad51 on double-stranded DNA. Proc. Natl Acad. Sci. USA 103, 1221–1226 (2006).
Tafvizi, A., Huang, F., Fersht, A. R., Mirny, L. A. & van Oijen, A. M. Tumor suppressor p53 slides on DNA with low friction and high stability. Biophys. J. 95, L01–L03 (2008).
Kim, J. H. & Larson, R. G. Single-molecule analysis of 1D diffusion and transcription elongation of T7 RNA polymerase along individual stretched DNA molecules. Nucleic Acids Res. 35, 3848–3858 (2007).
Schurr, J. M. The one-dimensional diffusion coefficient of proteins absorbed on DNA. Hydrodynamic considerations. Biophys. Chem. 9, 413–414 (1979).
Blainey, P. C. et al. Nonspecifically bound proteins spin while diffusing along DNA. Nature Struct. Mol. Biol. 16, 1224–1229 (2009).
Li, G. W., Berg, O. G. & Elf, J. Effects of macromolecular crowding and DNA looping on gene regulation kinetics. Nature Phys. 5, 294–297 (2009).
Stanford, N. P., Szczelkun, M. D., Marko, J. F. & Halford, S. E. One- and three-dimensional pathways for proteins to reach specific DNA sites. EMBO J. 19, 6546–6557 (2000).
Winter, R. B., Berg, O. G. & von Hippel, P. H. Diffusion-driven mechanisms of protein translocation on nucleic acids. 3. The Escherichia coli lac repressor–operator interaction: kinetic measurements and conclusions. Biochemistry 20, 6961–6977 (1981).
Gowers, D. M. & Halford, S. E. Protein motion from non-specific to specific DNA by three-dimensional routes aided by supercoiling. EMBO J. 22, 1410–1418 (2003).
Bonnet, I. et al. Sliding and jumping of single EcoRV restriction enzymes on non-cognate DNA. Nucleic Acids Res. 36, 4118–4127 (2008).
Porecha, R. H. & Stivers, J. T. Uracil DNA glycosylase uses DNA hopping and short-range sliding to trap extrahelical uracils. Proc. Natl Acad. Sci. USA 105, 10791–10796 (2008).
Golding, I., Paulsson, J., Zawilski, S. M. & Cox, E. C. Real-time kinetics of gene activity in individual bacteria. Cell 123, 1025–1036 (2005). This article uses MS2–GFP to follow the production of individual mRNA molecules in real time in bacterial cells.
Le, T. T. et al. Real-time RNA profiling within a single bacterium. Proc. Natl Acad. Sci. USA 102, 9160–9164 (2005).
Chubb, J. R., Trcek, T., Shenoy, S. M. & Singer, R. H. Transcriptional pulsing of a developmental gene. Curr. Biol. 16, 1018–1025 (2006).
Raj, A., Peskin, C. S., Tranchina, D., Vargas, D. Y. & Tyagi, S. Stochastic mRNA synthesis in mammalian cells. PLoS Biol. 4, e309 (2006).
Cai, L., Friedman, N. & Xie, X. S. Stochastic protein expression in individual cells at the single molecule level. Nature 440, 358–362 (2006).
Nagai, T. et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nature Biotechnol. 20, 87–90 (2002).
Berg, O. G. A model for the statistical fluctuations of protein numbers in a microbial population. J. Theor. Biol. 71, 587–603 (1978).
Rigney, D. R. Stochastic-model of constitutive protein levels in growing and dividing bacterial cells. J. Theor. Biol. 76, 453–480 (1979).
McAdams, H. H. & Arkin, A. Stochastic mechanisms in gene expression. Proc. Natl Acad. Sci. USA 94, 814–819 (1997).
Choi, P. J., Cai, L., Frieda, K. & Xie, X. S. A stochastic single-molecule event triggers phenotype switching of a bacterial cell. Science 322, 442–446 (2008). This article investigates the triggering mechanism of the lac operon bistable switch and shows that the dissociation of a single lac repressor is responsible for the phenotype switch to the induced state.
Tyagi, S. Imaging intracellular RNA distribution and dynamics in living cells. Nature Methods 6, 331–338 (2009).
Bertrand, E. et al. Localization of ASH1 mRNA particles in living yeast. Mol. Cell 2, 437–445 (1998). This study develops an approach to image individual mRNA molecules in living cells, using fluorescent protein-fused MS2 proteins that bind to an engineered mRNA sequence.
Beach, D. L., Salmon, E. D. & Bloom, K. Localization and anchoring of mRNA in budding yeast. Curr. Biol. 9, 569–578 (1999).
Johansson, H. E. et al. A thermodynamic analysis of the sequence-specific binding of RNA by bacteriophage MS2 coat protein. Proc. Natl Acad. Sci. USA 95, 9244–9249 (1998).
Delbrück, M. Statistical fluctuations in autocatalytic reactions. J. Chem. Phys. 8, 120–140 (1940).
Paulsson, J. Summing up the noise in gene networks. Nature 427, 415–418 (2004).
Shahrezaei, V. & Swain, P. S. Analytical distributions for stochastic gene expression. Proc. Natl Acad. Sci. USA 105, 17256–17261 (2008).
Gillespie, D. T. A general method for numerically simulating the stochastic time evolution of coupled chemical reactions. J. Comput. Phys. 22, 403–434 (1976).
Paulsson, J. & Ehrenberg, M. Random signal fluctuations can reduce random fluctuations in regulated components of chemical regulatory networks. Phys. Rev. Lett. 84, 5447–5450 (2000).
Friedman, N., Cai, L. & Xie, X. S. Linking stochastic dynamics to population distribution: an analytical framework of gene expression. Phys. Rev. Lett. 97, 168302 (2006). This article derives gamma distribution of protein copy numbers in a model with burst-like gene expression; the distribution is in agreement with experimental observations in reference 62.
Huh, W. K. et al. Global analysis of protein localization in budding yeast. Nature 425, 686–691 (2003).
Newman, J. R. et al. Single-cell proteomic analysis of S. cerevisiae reveals the architecture of biological noise. Nature 441, 840–846 (2006).
Bar-Even, A. et al. Noise in protein expression scales with natural protein abundance. Nature Genet. 38, 636–643 (2006).
Swain, P. S., Elowitz, M. B. & Siggia, E. D. Intrinsic and extrinsic contributions to stochasticity in gene expression. Proc. Natl Acad. Sci. USA 99, 12795–12800 (2002).
Thattai, M. & van Oudenaarden, A. Intrinsic noise in gene regulatory networks. Proc. Natl Acad. Sci. USA 98, 8614–8619 (2001).
Elowitz, M. B., Levine, A. J., Siggia, E. D. & Swain, P. S. Stochastic gene expression in a single cell. Science 297, 1183–1186 (2002).
Femino, A. M., Fay, F. S., Fogarty, K. & Singer, R. H. Visualization of single RNA transcripts in situ . Science 280, 585–590 (1998).
Maamar, H., Raj, A. & Dubnau, D. Noise in gene expression determines cell fate in Bacillus subtilis . Science 317, 526–529 (2007). This article used single-molecule FISH to show that the noise in the expression of a transcription factor determines whether a Bacillus subtilis cell becomes competent.
Jacob, F. & Monod, J. Genetic regulatory mechanisms in the synthesis of proteins. J. Mol. Biol. 3, 318–356 (1961).
Ozbudak, E. M., Thattai, M., Lim, H. N., Shraiman, B. I. & Van Oudenaarden, A. Multistability in the lactose utilization network of Escherichia coli . Nature 427, 737–740 (2004).
Novick, A. & Weiner, M. Enzyme induction as an all-or-none phenomenon. Proc. Natl Acad. Sci. USA 43, 553–566 (1957).
Dubnau, D. & Losick, R. Bistability in bacteria. Mol. Microbiol. 61, 564–572 (2006).
Balaban, N. Q., Merrin, J., Chait, R., Kowalik, L. & Leibler, S. Bacterial persistence as a phenotypic switch. Science 305, 1622–1625 (2004).
Ptashne, M. A Genetic Switch 3rd edn (Cold Spring Harbor Laboratory Press, 2004).
Losick, R. & Desplan, C. Stochasticity and cell fate. Science 320, 65–68 (2008).
Hanna, J. et al. Direct cell reprogramming is a stochastic process amenable to acceleration. Nature 462, 595–601 (2009).
Acknowledgements
We thank L. Cai, J. Xiao, J. Yu, N. Friedman, J. Elf, P. Choi, H. Chen and Y. Taniguchi for their key contributions to the work described, and W. Greenleaf, P. Sims and H. Ge for their helpful comments on this review. This work is supported by the National Institutes of Health Director's Pioneer Award and the Bill & Melinda Gates Foundation.
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Li, GW., Xie, X. Central dogma at the single-molecule level in living cells. Nature 475, 308–315 (2011). https://doi.org/10.1038/nature10315
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