Central dogma at the single-molecule level in living cells (original) (raw)

• 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.
  ADS CAS PubMed Google Scholar
• 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).
  ADS CAS Google Scholar
• Betzig, E. & Chichester, R. J. Single molecules observed by near-field scanning optical microscopy. Science 262, 1422–1425 (1993).
  ADS CAS PubMed Google Scholar
• 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).
  ADS CAS PubMed Google Scholar
• 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).
  CAS Google Scholar
• Xie, X. S. & Trautman, J. K. Optical studies of single molecules at room temperature. Annu. Rev. Phys. Chem. 49, 441–480 (1998).
  ADS CAS PubMed Google Scholar
• Moerner, W. E. & Orrit, M. Illuminating single molecules in condensed matter. Science 283, 1670–1676 (1999).
  ADS CAS PubMed Google Scholar
• Ha, T. et al. Single-molecule fluorescence spectroscopy of enzyme conformational dynamics and cleavage mechanism. Proc. Natl Acad. Sci. USA 96, 893–898 (1999).
  ADS CAS PubMed PubMed Central Google Scholar
• 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.
  ADS CAS PubMed Google Scholar
• 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).
  CAS PubMed Google Scholar
• 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.
  ADS CAS PubMed Google Scholar
• 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).
  ADS CAS PubMed Google Scholar
• Bremer, H. A stochastic process determines the time at which cell division begins in Escherichia coli . J. Theor. Biol. 118, 351–365 (1986).
  CAS PubMed Google Scholar
• Wang, M. D. et al. Force and velocity measured for single molecules of RNA polymerase. Science 282, 902–907 (1998).
  ADS CAS PubMed Google Scholar
• 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).
  ADS CAS PubMed PubMed Central Google Scholar
• Kapanidis, A. N. et al. Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism. Science 314, 1144–1147 (2006).
  ADS PubMed PubMed Central Google Scholar
• Wen, J. D. et al. Following translation by single ribosomes one codon at a time. Nature 452, 598–603 (2008).
  ADS CAS PubMed PubMed Central Google Scholar
• Cornish, P. V., Ermolenko, D. N., Noller, H. F. & Ha, T. Spontaneous intersubunit rotation in single ribosomes. Mol. Cell 30, 578–588 (2008).
  CAS PubMed PubMed Central Google Scholar
• 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).
  ADS CAS PubMed PubMed Central Google Scholar
• Ishihama, Y. et al. Protein abundance profiling of the Escherichia coli cytosol. BMC Genomics 9, 102 (2008).
  PubMed PubMed Central Google Scholar
• 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.
  ADS CAS PubMed PubMed Central Google Scholar
• 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).
  CAS PubMed Google Scholar
• 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).
  ADS CAS PubMed Google Scholar
• Andersson, H., Baechi, T., Hoechl, M. & Richter, C. Autofluorescence of living cells. J. Microsc. 191, 1–7 (1998).
  CAS PubMed Google Scholar
• 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).
  CAS PubMed Google Scholar
• 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).
  CAS PubMed PubMed Central Google Scholar
• Wilson, T. Confocal Microscopy (Academic, 1990).
  Google Scholar
• Denk, W., Strickler, J. H. & Webb, W. W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).
  ADS CAS PubMed Google Scholar
• 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).
  CAS Google Scholar
• Fuchs, E., Jaffe, J., Long, R. & Azam, F. Thin laser light sheet microscope for microbial oceanography. Opt. Express 10, 145–154 (2002).
  ADS PubMed Google Scholar
• 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).
  ADS PubMed PubMed Central Google Scholar
• 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).
  CAS PubMed PubMed Central Google Scholar
• Xie, X. S., Yu, J. & Yang, W. Y. Living cells as test tubes. Science 312, 228–230 (2006).
  ADS CAS PubMed Google Scholar
• 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.
  ADS CAS PubMed Google Scholar
• 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.
  ADS CAS PubMed PubMed Central Google Scholar
• Thompson, R. E., Larson, D. R. & Webb, W. W. Precise nanometer localization analysis for individual fluorescent probes. Biophys. J. 82, 2775–2783 (2002).
  CAS PubMed PubMed Central Google Scholar
• Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nature Methods 3, 793–795 (2006).
  CAS PubMed PubMed Central Google Scholar
• Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).
  ADS CAS PubMed Google Scholar
• Hess, S. T., Girirajan, T. P. & Mason, M. D. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys. J. 91, 4258–4272 (2006).
  ADS CAS PubMed PubMed Central Google Scholar
• Hell, S. W. Far-field optical nanoscopy. Science 316, 1153–1158 (2007).
  ADS CAS PubMed Google Scholar
• 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).
  CAS PubMed Google Scholar
• 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).
  CAS PubMed Google Scholar
• Riggs, A. D., Bourgeois, S. & Cohn, M. The lac repressor–operator interaction. 3. Kinetic studies. J. Mol. Biol. 53, 401–417 (1970).
  CAS PubMed Google Scholar
• Kabata, H. et al. Visualization of single molecules of RNA polymerase sliding along DNA. Science 262, 1561–1563 (1993).
  ADS CAS PubMed Google Scholar
• Harada, Y. et al. Single-molecule imaging of RNA polymerase-DNA interactions in real time. Biophys. J. 76, 709–715 (1999).
  ADS CAS PubMed PubMed Central Google Scholar
• 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).
  ADS CAS PubMed PubMed Central Google Scholar
• 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).
  ADS CAS PubMed PubMed Central Google Scholar
• 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).
  CAS PubMed PubMed Central Google Scholar
• 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).
  CAS PubMed PubMed Central Google Scholar
• Schurr, J. M. The one-dimensional diffusion coefficient of proteins absorbed on DNA. Hydrodynamic considerations. Biophys. Chem. 9, 413–414 (1979).
  CAS PubMed Google Scholar
• Blainey, P. C. et al. Nonspecifically bound proteins spin while diffusing along DNA. Nature Struct. Mol. Biol. 16, 1224–1229 (2009).
  CAS Google Scholar
• 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).
  ADS CAS Google Scholar
• 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).
  CAS PubMed PubMed Central Google Scholar
• 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).
  CAS PubMed Google Scholar
• 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).
  CAS PubMed PubMed Central Google Scholar
• Bonnet, I. et al. Sliding and jumping of single EcoRV restriction enzymes on non-cognate DNA. Nucleic Acids Res. 36, 4118–4127 (2008).
  ADS CAS PubMed PubMed Central Google Scholar
• 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).
  ADS CAS PubMed PubMed Central Google Scholar
• 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.
  CAS PubMed Google Scholar
• Le, T. T. et al. Real-time RNA profiling within a single bacterium. Proc. Natl Acad. Sci. USA 102, 9160–9164 (2005).
  ADS CAS PubMed PubMed Central Google Scholar
• Chubb, J. R., Trcek, T., Shenoy, S. M. & Singer, R. H. Transcriptional pulsing of a developmental gene. Curr. Biol. 16, 1018–1025 (2006).
  CAS PubMed PubMed Central Google Scholar
• Raj, A., Peskin, C. S., Tranchina, D., Vargas, D. Y. & Tyagi, S. Stochastic mRNA synthesis in mammalian cells. PLoS Biol. 4, e309 (2006).
  PubMed PubMed Central Google Scholar
• Cai, L., Friedman, N. & Xie, X. S. Stochastic protein expression in individual cells at the single molecule level. Nature 440, 358–362 (2006).
  ADS CAS PubMed Google Scholar
• 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).
  CAS Google Scholar
• Berg, O. G. A model for the statistical fluctuations of protein numbers in a microbial population. J. Theor. Biol. 71, 587–603 (1978).
  CAS PubMed Google Scholar
• Rigney, D. R. Stochastic-model of constitutive protein levels in growing and dividing bacterial cells. J. Theor. Biol. 76, 453–480 (1979).
  MathSciNet CAS PubMed Google Scholar
• McAdams, H. H. & Arkin, A. Stochastic mechanisms in gene expression. Proc. Natl Acad. Sci. USA 94, 814–819 (1997).
  ADS CAS PubMed PubMed Central Google Scholar
• 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.
  ADS CAS PubMed PubMed Central Google Scholar
• Tyagi, S. Imaging intracellular RNA distribution and dynamics in living cells. Nature Methods 6, 331–338 (2009).
  CAS PubMed Google Scholar
• 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.
  CAS PubMed Google Scholar
• Beach, D. L., Salmon, E. D. & Bloom, K. Localization and anchoring of mRNA in budding yeast. Curr. Biol. 9, 569–578 (1999).
  CAS PubMed Google Scholar
• 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).
  ADS CAS PubMed PubMed Central Google Scholar
• Delbrück, M. Statistical fluctuations in autocatalytic reactions. J. Chem. Phys. 8, 120–140 (1940).
  ADS Google Scholar
• Paulsson, J. Summing up the noise in gene networks. Nature 427, 415–418 (2004).
  ADS CAS PubMed Google Scholar
• Shahrezaei, V. & Swain, P. S. Analytical distributions for stochastic gene expression. Proc. Natl Acad. Sci. USA 105, 17256–17261 (2008).
  ADS CAS PubMed PubMed Central Google Scholar
• Gillespie, D. T. A general method for numerically simulating the stochastic time evolution of coupled chemical reactions. J. Comput. Phys. 22, 403–434 (1976).
  ADS MathSciNet CAS Google Scholar
• 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).
  ADS CAS PubMed Google Scholar
• 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.
  ADS PubMed Google Scholar
• Huh, W. K. et al. Global analysis of protein localization in budding yeast. Nature 425, 686–691 (2003).
  ADS CAS PubMed Google Scholar
• Newman, J. R. et al. Single-cell proteomic analysis of S. cerevisiae reveals the architecture of biological noise. Nature 441, 840–846 (2006).
  ADS CAS PubMed Google Scholar
• Bar-Even, A. et al. Noise in protein expression scales with natural protein abundance. Nature Genet. 38, 636–643 (2006).
  CAS PubMed Google Scholar
• 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).
  ADS CAS PubMed PubMed Central Google Scholar
• Thattai, M. & van Oudenaarden, A. Intrinsic noise in gene regulatory networks. Proc. Natl Acad. Sci. USA 98, 8614–8619 (2001).
  ADS CAS PubMed PubMed Central Google Scholar
• Elowitz, M. B., Levine, A. J., Siggia, E. D. & Swain, P. S. Stochastic gene expression in a single cell. Science 297, 1183–1186 (2002).
  ADS CAS PubMed Google Scholar
• Femino, A. M., Fay, F. S., Fogarty, K. & Singer, R. H. Visualization of single RNA transcripts in situ . Science 280, 585–590 (1998).
  ADS CAS PubMed Google Scholar
• 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.
  ADS CAS PubMed Google Scholar
• Jacob, F. & Monod, J. Genetic regulatory mechanisms in the synthesis of proteins. J. Mol. Biol. 3, 318–356 (1961).
  CAS PubMed Google Scholar
• 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).
  ADS CAS PubMed Google Scholar
• Novick, A. & Weiner, M. Enzyme induction as an all-or-none phenomenon. Proc. Natl Acad. Sci. USA 43, 553–566 (1957).
  ADS CAS PubMed PubMed Central Google Scholar
• Dubnau, D. & Losick, R. Bistability in bacteria. Mol. Microbiol. 61, 564–572 (2006).
  CAS PubMed Google Scholar
• Balaban, N. Q., Merrin, J., Chait, R., Kowalik, L. & Leibler, S. Bacterial persistence as a phenotypic switch. Science 305, 1622–1625 (2004).
  ADS CAS PubMed Google Scholar
• Ptashne, M. A Genetic Switch 3rd edn (Cold Spring Harbor Laboratory Press, 2004).
  Google Scholar
• Losick, R. & Desplan, C. Stochasticity and cell fate. Science 320, 65–68 (2008).
  ADS CAS PubMed PubMed Central Google Scholar
• Hanna, J. et al. Direct cell reprogramming is a stochastic process amenable to acceleration. Nature 462, 595–601 (2009).
  ADS CAS PubMed PubMed Central Google Scholar