Cell-to-cell diversity in protein levels of a gene driven by a tetracycline inducible promoter
Tóm tắt
Gene expression in Escherichia coli is regulated by several mechanisms. We measured in single cells the expression level of a single copy gene coding for green fluorescent protein (GFP), integrated into the genome and driven by a tetracycline inducible promoter, for varying induction strengths. Also, we measured the transcriptional activity of a tetracycline inducible promoter controlling the transcription of a RNA with 96 binding sites for MS2-GFP. The distribution of GFP levels in single cells is found to change significantly as induction reaches high levels, causing the Fano factor of the cells' protein levels to increase with mean level, beyond what would be expected from a Poisson-like process of RNA transcription. In agreement, the Fano factor of the cells' number of RNA molecules target for MS2-GFP follows a similar trend. The results provide evidence that the dynamics of the promoter complex formation, namely, the variability in its duration from one transcription event to the next, explains the change in the distribution of expression levels in the cell population with induction strength. The results suggest that the open complex formation of the tetracycline inducible promoter, in the regime of strong induction, affects significantly the dynamics of RNA production due to the variability of its duration from one event to the next.
Tài liệu tham khảo
Neubauerz Z, Calef E: Immunity phase-shift in defective lysogens: non-mutational hereditary change of early regulation of lambda prophage. Journal of Molecular Biology. 1970, 51: 1-13. 10.1016/0022-2836(70)90265-2
Arkin A, Ross J, McAdams HM: Stochastic kinetic analysis of developmental pathway bifurcation in phage λ-infected Escherichia coli cells. Genetics. 1998, 149: 1633-1648.
Acar M, Mettetal J, van Oudenaarden : A stochastic switching as a survival strategy in fluctuating environments. Nature Genetics. 2008, 40: 471-475. 10.1038/ng.110
Lee HH, Molla MN, Cantor CR, Collins JJ: Bacterial charity work leads to population-wide resistance. Nature. 2010, 467: 82-86. 10.1038/nature09354
von Hippel P, Bear D, Morgan W, McSwiggen J: Protein-nucleic acid interactions in transcription: A molecular analysis. Annual Review of Biochemistry. 1984, 53: 389- 10.1146/annurev.bi.53.070184.002133
McClure WR: Rate-limiting steps in RNA chain initiation. PNAS. 1980, 77: 5634-5638. 10.1073/pnas.77.10.5634
Yu J, Xiao J, Ren X, Lao K, Xie S: Probing gene expression in live cells, one protein molecule at a time. Science. 2006, 311: 1600-1603. 10.1126/science.1119623
Taniguchi Y, Choi PJ, Li GW, Chen H, Babu M, Hearn J, Emili A, Xie S: Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells. Science. 2001, 329: 533-538.
Kaern M, Elston TR, Blake WJ, Collins JJ: Stochasticity in gene expression. Nature Reviews Genetics. 2005, 6: 451-464. 10.1038/nrg1615
Hillen W, Berens C: Mechanisms underlying expression of Tn10 encoded tetracycline resistance. Annual Review of Microbiology. 1994, 48: 345-369. 10.1146/annurev.mi.48.100194.002021
McClure WR: Mechanism and control of transcription initiation in prokaryotes. Annual Review of Biochemistry. 1985, 54: 171-120. 10.1146/annurev.bi.54.070185.001131
Lutz R, Bujard H: Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Research. 1997, 25: 1203-1210. 10.1093/nar/25.6.1203
Ashcroft M, Kubbutat M, Vousden K: Regulation of p53 Function and Stability by Phosphorylation. Molecular Cell Biology. 1999, 19: 1751-1758.
Dai X, Healy S, Yli-Harja O, Ribeiro AS: Tuning cell differentiation patterns and single cell dynamics by regulating proteins' functionalities in a Toggle Switch. Journal of Theoretical Biology. 2009, 261 (3): 441-448. 10.1016/j.jtbi.2009.08.024
Buc H, McClure WR: Kinetics of open complex formation between Escherichia coli RNA polymerase and the lac UV5 promoter. Evidence for a sequential mechanism involving three steps. Biochemistry. 1985, 24: 2712-2723. 10.1021/bi00332a018
Lutz R, Lozinski T, Ellinger T, Bujard H: Dissecting the functional program of Escherichia coli promoters: the combined mode of action of Lac repressor and AraC activator. Nucleic Acids Research. 2001, 29: 3873-3881. 10.1093/nar/29.18.3873
Ribeiro AS, Häkkinen A, Mannerström H, Lloyd-Price J, Yli-Harja O: Effects of the promoter open complex formation on gene expression dynamics. Physical Review E. 2010, 81: 011912-
Ribeiro AS: Effects of coupling strength and space on the dynamics of coupled toggle switches in stochastic gene networks with multiple-delayed reactions. Physical Review E. 2007, 75: 061903-
Golding I, Paulsson J, Zawilski SM, Cox EC: Real-time kinetics of gene activity in individual bacteria. Cell. 2005, 123: 1025-1036. 10.1016/j.cell.2005.09.031
Le TT, Harlepp S, Guet CC, Dittmar K, Emonet T, Pan T, Cluzel P: Real-time RNA profiling within a single bacterium. Proc. Natl. Acad. Sci. U. S. A. 2005, 102: 9160-9164. 10.1073/pnas.0503311102.
Datsenko KA, Wanner BL: One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U. S. A. 2000, 97: 6640-6645. 10.1073/pnas.120163297
Andersen JB, Sternberg C, Poulsen LK, Bjørn SP, Givskov M, Molin S: New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Applied Environmental Microbiology. 1998, 64: 2240-2246.
Elowitz MB, Leibler S: A synthetic oscillatory network of transcriptional regulators. Nature. 2000, 403: 335-338. 10.1038/35002125
Skerra A: Use of the tetracycline promoter for the tightly regulated production of a murine antibody fragment in Escherichia coli. Gene. 1994, 151: 131-135. 10.1016/0378-1119(94)90643-2
Korpela MT, Kurittu JS, Karvinen JT, Karp MT: A recombinant Escherichia coli sensor strain for the detection of tetracyclines. Analytical Chemistry. 1998, 70: 4457-4462. 10.1021/ac980740e
Hansson MD, Rzeznicka K, Rosenbäck M, Hansson M, Sirijovski N: PCR-mediated deletion of plasmid DNA. Analytical Biochemistry. 2008, 15: 373-375.
Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning: A Laboratory Manual. 1982, New York: Cold Spring Harbor Laboratory, New York
Soille P: Morphological Image Analysis: Principles and Applications. 1999, Springer-Verlag
Chen TB, Lu H, Lee Y-S, Lan H-J: Segmentation of cDNA microarray images by kernel density estimation. Journal of Biomedical Informatics. 2008, 41: 1021-1027. 10.1016/j.jbi.2008.02.007
Otsu N: A threshold selection method from gray-level histograms. IEEE Transactions on Systems, Man, Cybernetics. 1979, 9: 62-66.
Ribeiro AS, Zhu R, Kauffman SA: A general modeling strategy for gene regulatory networks with stochastic dynamics. Journal of Computational Biology. 2007, 13: 1630-1639.
Roussel M, Zhu R: Validation of an algorithm for delay stochastic simulation of transcription and translation in prokaryotic gene expression. Physical Biology. 2006, 3: 274-284. 10.1088/1478-3975/3/4/005
Zhu R, Ribeiro S, Salahub D, Kauffman SA: Studying genetic regulatory networks at the molecular level: de-layed reaction stochastic models. Journal of Theoretical Biology. 2007, 246: 725-745. 10.1016/j.jtbi.2007.01.021
Ribeiro AS, Lloyd-Price J: SGNSim, a stochastic genetic networks simulator. Bioinformatics. 2007, 23: 777-779. 10.1093/bioinformatics/btm004
Gillespie DT: Exact stochastic simulation of coupled chemical reactions. Journal of Physical Chemistry. 1977, 81: 2340-2361. 10.1021/j100540a008.
Ribeiro AS, Smolander OP, Rajala T, Häkkinen A, Yli-Harja O: Delayed stochastic model of transcription at the single nucleotide level. Journal of Computational Biology. 2009, 16: 539-553. 10.1089/cmb.2008.0153
Kleinschmidt C, Tovar K, Hillen W, Porschke D: Dynamics of repressor-operator recognition: the Tn10-encoded tetracycline resistance control. Biochemistry. 1988, 27: 1094-104. 10.1021/bi00404a003
Kamionka A, Bogdanska-Urbaniak J, Scholz O, Hillen W: Two mutations in the tetracycline repressor change the inducer anhydrotetracycline to a corepressor. Nucleic Acids Research. 2004, 32: 842-847. 10.1093/nar/gkh200
Cormack BP, Valdivia RH, Falkow S: FACS-optimized mutants of the green fluorescent protein (GFP). Gene. 1996, 173: 33-38. 10.1016/0378-1119(95)00685-0
Zhu R, Salahub D: Delay stochastic simulation of single-gene expression reveals a detailed relationship between protein noise and mean abundance. FEBS Letters. 2008, 582: 2905-291. 10.1016/j.febslet.2008.07.028
Paulsson J: Summing up the noise in gene networks. Nature. 2004, 29: 415-418.
Landick R: The regulatory roles and mechanism of transcriptional pausing. Biochemical Society Transactions. 2006, 34: 1062-1066.
Bar-Even A, Paulsson J, Maheshri N, Carmi M, O'Shea E, Pilpel Y, Barkai N: Noise in protein expression scales with natural protein abundance. Nature Genetics. 2006, 38: 636-643. 10.1038/ng1807
Ribeiro AS: Stochastic and delayed stochastic models of gene expression and regulation. Mathematical Biosciences. 2010, 223: 1-11. 10.1016/j.mbs.2009.10.007
Greive SJ, von Hippel PH: Thinking quantitatively about transcriptional regulation. Nature Reviews Molecular Cell Biology. 2005, 6: 221-32.