RNA聚合酶动态调控DNA转录的单分子水平研究进展

doi:10.3969/j.issn.0253-9608.2022.05.010

摘要/Abstract

摘要： 真核生物的RNA聚合酶II(Pol II)和原核生物的RNA聚合酶(RNAP)主要负责转录合成信使RNA(mRNA)，调控不同基因的转录水平，以调节生物体的生长发育和应对复杂多变的环境。研究者采用传统的荧光显微镜观测到RNAP可形成团簇，据此针对DNA转录调控提出“转录工厂”模型。随着单分子技术的发展，研究者在单分子水平上观测到了活细胞中RNAP动态调控DNA转录，提出RNAP可以通过液-液相分离机制进行转录调控。该综述总结了不同单分子荧光显微镜的技术原理，以及相关的荧光探针标记方法，并介绍了在真核生物和原核生物中应用单分子成像技术，可视化RNA聚合酶动态调控DNA转录过程的研究进展，最后展望了单分子技术在转录调控研究中的应用前景。

关键词: RNA聚合酶, 动态调控, DNA转录, 单分子成像, 相分离

Abstract: Eukaryotic RNA polymerase II (Pol II) and prokaryotic RNA polymerase (RNAP) are mainly dedicated to messenger RNA (mRNA) synthesis, influencing the growth and development of organisms as well as in response to complicated environment conditions by regulating the transcriptional level of different genes. Clusters assembled by RNA polymerases were observed using conventional fluorescence microscopy, which was proposed as a “transcription factories” model for DNA transcription regulation. However, along with the development of single-molecule technology, dynamic regulation of transcription by RNAP was observed at single-molecule level, which thus raised the liquid-liquid phase separation model of RNAP transcription regulation. This paper reviewed the technical principles of multiple single-molecule fluorescent microscopies and the related labeling strategies via fluorescence probes. The advances in application of single-molecule technology in visualizing dynamic DNA transcription regulation of RNA polymerase were presented for both prokaryotes and eukaryotes. The application prospects of transcription regulation investigation using single-molecule technologies are introduced briefly at the end of this paper.

郝理, 江婷, 樊军. RNA聚合酶动态调控DNA转录的单分子水平研究进展[J]. 自然杂志, 2023, 45(1): 33-44.

HAO Li, JIANG Ting, FAN Jun. Advances in single-molecule investigation of dynamic DNA transcription regulation by RNA polymerase[J]. Chinese Journal of Nature, 2023, 45(1): 33-44.

参考文献

[1] SHANDILYA J, ROBERTS S G. The transcription cycle in eukaryotes: from productive initiation to RNA polymerase II recycling [J]. Biochimica et Biophysica Acta, 2012, 1819(5): 391-400.
[2] CRAMER P. Organization and regulation of gene transcription [J]. Nature, 2019, 573(7772): 45-54.
[3] CHEN J, BOYACI H, CAMPBELL E A. Diverse and unified mechanisms of transcription initiation in bacteria [J]. Nature Reviews Microbiology, 2021, 19(2): 95-109.
[4] BROWNING D F, BUSBY S J. Local and global regulation of transcription initiation in bacteria [J]. Nature Reviews Microbiology,
2016, 14(10): 638-650.
[5] ROEDER R G. 50+ years of eukaryotic transcription: an expanding universe of factors and mechanisms [J]. Nature Structural &
Molecular Biology, 2019, 26(9): 783-791.
[6] WERNER F, GROHMANN D. Evolution of multisubunit RNA polymerases in the three domains of life [J]. Nature Reviews
Microbiology, 2011, 9(2): 85-98.
[7] LIU X, BUSHNELL D A, KORNBERG R D. RNA polymerase II transcription: structure and mechanism [J]. Biochimica et
Biophysica Acta, 2013, 1829(1): 2-8.
[8] LOUDER R K, HE Y, LOPEZ-BLANCE J R, et al. Structure of promoter-bound TFIID and model of human pre-initiation complex
assembly [J]. Nature, 2016, 531(7596): 604-609.
[9] JOHNSON D S, MORTAZAVI A, MYERS R M, et al. Genomewide mapping of in vivo protein-DNA interactions [J]. Science,
2007, 316(5830): 1497-1502.
[10] RHEE H S, PUGH B F. Comprehensive genome-wide protein-DNA interactions detected at single-nucleotide resolution [J]. Cell, 2011, 147(6): 1408-1419.
[11] ZHANG Z, TJIAN R. Measuring dynamics of eukaryotic transcription initiation: challenges, insights and opportunities [J].
Transcription, 2018, 9(3): 159-165.
[12] HOBOTH P, SEBESTA O, HOZAK P. How single-molecule localization microscopy expanded our mechanistic understanding
of RNA polymerase II transcription [J]. International Journal of Molecular Sciences, 2021, 22(13): 6694.
[13] LI G W, XIE X S. Central dogma at the single-molecule level in living cells [J]. Nature, 2011, 475(7356): 308-315.
[14] LIU Z, LAVIS L D, BETZIG E. Imaging live-cell dynamics and structure at the single-molecule level [J]. Molecular Cell, 2015,
58(4): 644-659.
[15] WANG Z H, DENG W L. Dynamic transcription regulation at the single-molecule level [J]. Developmental Biology, 2022, 482: 67-81.
[16] KREUTZBERGER A J B, JI M, AARON J, et al. Rhomboid distorts lipids to break the viscosity-imposed speed limit of membrane
diffusion [J]. Science, 2019, 363(6426): eaao0076.
[17] WANG X H, LI X J, DENG X, et al. Single-molecule fluorescence imaging to quantify membrane protein dynamics and oligomerization in living plant cells [J]. Nature Protocols, 2015, 10(12): 2054-2063.
[18] CUI Y, YU M, YAO X, et al. Single-particle tracking for the quantification of membrane protein dynamics in living plant cells
[J]. Molecular Plant, 2018, 11(11): 1315-1327.
[19] TOKUNAGA M, IMAMOTO N, SAKATA-SOGAWA K. Highly inclined thin illumination enables clear single-molecule imaging in
cells [J]. Nature Methods, 2008, 5(2): 159-161.
[20] GEBHARDT J C, SUTER D M, ROY R, et al. Single-molecule imaging of transcription factor binding to DNA in live mammalian
cells [J]. Nature Methods, 2013, 10(5): 421-426.
[21] CHEN B C, LEGANT W R, WANG K, et al. Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal
resolution [J]. Science, 2014, 346(6208): 1257998.
[22] SHIMOMURA O, JOHNSON F H, SAIGA Y. Extraction, purification and properties of aequorin, a bioluminescent protein
from the luminous hydromedusan, Aequorea [J]. Journal of Cellular and Comparative Physiology, 1962, 59: 223-239.
[23] 邢晶晶, 林金星. 活细胞单分子荧光标记——点亮生命微观世界的繁星[J]. 生命世界, 2015(12): 48-53.
[24] WANG S, MOFFITT J R, DEMPSEY G T, et al. Characterization and development of photoactivatable fluorescent proteins for singlemolecule-based superresolution imaging [J]. Proceedings of the National Academy of Sciences of the United States of America, 2014, 111(23): 8452-8457.
[25] XIA T, LI N, FANG X. Single-molecule fluorescence imaging in living cells [J]. Annual Review of Physical Chemistry, 2013, 64:
459-480.
[26] FERNANDEZ-SUAREZ M, TING A Y. Fluorescent probes for super-resolution imaging in living cells [J]. Nature Reviews
Molecular Cell Biology, 2008, 9(12): 929-943.
[27] BETZIG E, PATTERSON G H, SOUGRAT R, et al. Imaging intracellular fluorescent proteins at nanometer resolution [J].
Science, 2006, 313(5793): 1642-1645.
[28] LIPPINCOTT-SCHWARTZ J, PATTERSON G H. Photoactivatable fluorescent proteins for diffraction-limited and super-resolution
imaging [J]. Trends in Cell Biology, 2009, 19(11): 555-565.
[29] GRIMM J B, ENGLISH B P, CHEN J, et al. A general method to improve fluorophores for live-cell and single-molecule microscopy [J]. Nature Methods, 2015, 12(3): 244-250.
[30] ZHANG M, CHANG H, ZHANG Y, et al. Rational design of true monomeric and bright photoactivatable fluorescent proteins [J].
Nature Methods, 2012, 9(7): 727-729.
[31] LANDGRAF D, OKUMUS B, CHIEN P, et al. Segregation of molecules at cell division reveals native protein localization [J].
Nature Methods, 2012, 9(5): 480-482.
[32] LOS G V, ENCELL L P, MCDOUGALL M G, et al. HaloTag: a novel protein labeling technology for cell imaging and protein
analysis [J]. ACS Chemical Biology, 2008, 3(6): 373-382.
[33] GAUTIER A, JUILLERAT A, HEINIS C, et al. An engineered protein tag for multiprotein labeling in living cells [J]. Chemistry &
Biology, 2008, 15(2): 128-136.
[34] CHEN Z, JING C, GALLAGHER S S, et al. Second-generation covalent TMP-tag for live cell imaging [J]. Journal of the American
Chemical Society, 2012, 134(33): 13692-13699.
[35] LAVIS L D. Teaching old dyes new tricks: biological probes built from fluoresceins and rhodamines [J]. Annual Review of
Biochemistry, 2017, 86: 825-843.
[36] GRIMM J B, ENGLISH B P, CHOI H, et al. Bright photoactivatable fluorophores for single-molecule imaging [J]. Nature Methods, 2016, 13(12): 985-988.
[37] BERTRAND E, CHARTRAND P, SCHAEFER M, et al. Localization of ASH1 mRNA particles in living yeast [J]. Molecular
Cell, 1998, 2(4): 437-445.
[38] CHAO J A, PATSKOVSKY Y, ALMO S C, et al. Structural basis for the coevolution of a viral RNA-protein complex [J]. Nature
Structural & Molecular Biology, 2008, 15(1): 103-105.
[39] HOCINE S, RAYMOND P, ZENKLUSEN D, et al. Single-molecule analysis of gene expression using two-color RNA labeling in live
yeast [J]. Nature Methods, 2013, 10(2): 119-121.
[40] 常振仪, 严维, 刘东风, 等. CRISPR/Cas技术研究进展[J]. 农业生物技术学报, 2015, 23(9): 1196-1206.
[41] YANG L Z, WANG Y, LI S Q, et al. Dynamic imaging of RNA in living cells by CRISPR-Cas13 systems [J]. Molecular Cell, 2019,
76(6): 981-997.e7.
[42] OUELLET J. RNA fluorescence with light-up aptamers [J]. Frontiers in Chemistry, 2016, 4: 29.
[43] TRACHMAN R J 3RD, AUTOUR A, JENG S C Y, et al. Structure and functional reselection of the Mango-III fluorogenic RNA
aptamer [J]. Nature Chemical Biology, 2019, 15(5): 472-479.
[44] 周子琦, 张洋子, 兰欣悦, 等. 发光核酸适配体的筛选及应用[J]. 生物技术通报, 2022, 38(5): 240-247.
[45] LIONNET T, WU C. Single-molecule tracking of transcription protein dynamics in living cells: Seeing is believing, but what are
we seeing? [J]. Current Opinion in Genetics & Development, 2021, 67, 94-102.
[46] RUST M J, BATES M, ZHUANG X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM) [J].
Nature Methods, 2006, 3(10): 793-795.
[47] HELL S W, WICHMANN J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion
fluorescence microscopy [J]. Optics Letters, 1994, 19(11): 780-782.
[48] GUSTAFSSON M G L. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy [J]. Journal of Microscopy, 2000, 198: 82-87.
[49] SMALL A, STAHLHEBER S. Fluorophore localization algorithms for super-resolution microscopy [J]. Nature Methods, 2014, 11(3): 267-279.
[50] JAQAMAN K, LOERKE D, METTLEN M, et al. Robust singleparticle tracking in live-cell time-lapse sequences [J]. Nature
Methods, 2008, 5(8): 695-702.
[51] CARRERO G, MCDONALD D, CRAWFORD E, et al. Using FRAP and mathematical modeling to determine the in vivo kinetics
of nuclear proteins [J]. Methods, 2003, 29(1): 14-28.
[52] DIGMAN M A, GRATTON E. Lessons in fluctuation correlation spectroscopy [J]. Annual Review of Physical Chemistry, 2011, 62:
645-668.
[53] 曲绍峰, 林金星, 李晓娟. FCS/FCCS技术及其在植物细胞生物学中的应用[J]. 电子显微学报, 2014, 33(5): 461-468.
[54] MICHELMAN-RIBEIRO A, MAZZA D, ROSALES T, et al. Direct measurement of association and dissociation rates of DNA
binding in live cells by fluorescence correlation spectroscopy [J]. Biophysical Journal, 2009, 97(1): 337-346.
[55] WHITE M D, ANGIOLINI J F, ALVAREZ Y D, et al. Long-lived binding of Sox2 to DNA predicts cell fate in the four-cell mouse
embryo [J]. Cell, 2016, 165(1): 75-87.
[56] BERLAND K M, SO P T, GRATTON E. Two-photon fluorescence correlation spectroscopy: method and application to the intracellular environment [J]. Biophysical Journal, 1995, 68(2): 694-701.
[57] SCHIER A C, TAATJES D J. Structure and mechanism of the RNA polymerase II transcription machinery [J]. Genes & Development, 2020, 34(7/8): 465-488.
[58] VERA M, BISWAS J, SENECAL A, et al. Single-cell and singlemolecule analysis of gene expression regulation [J]. Annual Review of Genetics, 2016, 50: 267-291.
[59] MUELLER F, STASEVICH T J, MAZZA D, et al. Quantifying transcription factor kinetics: at work or at play? [J]. Critical Reviews
in Biochemistry and Molecular Biology, 2013, 48(5): 492-514.
[60] HAGER G L, MCNALLY J G, MISTELI T. Transcription dynamics [J]. Molecular Cell, 2009, 35(6): 741-753.
[61] BROUWER I, LENSTRA T L. Visualizing transcription: key to understanding gene expression dynamics [J]. Current Opinion in
Chemical Biology, 2019, 51: 122-129.
[62] COOK P R. The organization of replication and transcription [J]. Science, 1999, 284(5421): 1790-1795.
[63] DARZACQ X, SHAV-TAL Y, DE TURRIS V, et al. In vivo dynamics of RNA polymerase II transcription [J]. Nature Structural
& Molecular Biology, 2007, 14(9): 796-806.
[64] ZHAO Z W, ROY R, GEBHARDT J C, et al. Spatial organization of RNA polymerase II inside a mammalian cell nucleus revealed by reflected light-sheet superresolution microscopy [J]. Proceedings of the National Academy of Sciences of the United States of America, 2014, 111 (2): 681-686.
[65] CISSE I I, IZEDDIN I, CAUSSE S Z, et al. Real-time dynamics of RNA polymerase II clustering in live human cells [J]. Science, 2013, 341(6146): 664-667.
[66] CHO W K, JAYANTH N, ENGLISH B P, et al. RNA Polymerase II cluster dynamics predict mRNA output in living cells [J]. Elife,
2016, 5: e13617.
[67] LI J R, DONG A K, SAYDAMINOVA K, et al. Single-molecule nanoscopy elucidates RNA polymerase II transcription at single
genes in live cells [J]. Cell, 2019, 178(2): 491-506.e28.
[68] NGUYEN V Q, RANJAN A, LIU S, et al. Spatiotemporal coordination of transcription preinitiation complex assembly in live
cells [J]. Molecular Cell, 2021, 81(17): 3560-3575.e6.
[69] BOEYNAEMS S, ALBERTI S, FAWZI N L, et al. Protein phase separation: a new phase in cell biology [J]. Trends in Cell Biology,
2018, 28(6): 420-435.
[70] HYMAN A A, WEBER C A, JULICHER F. Liquid-liquid phase separation in biology [J]. Annual Review of Cell and Developmental
Biology, 2014, 30: 39-58.
[71] LI P, BANJADE S, CHENG H C, et al. Phase transitions in the assembly of multivalent signalling proteins [J]. Nature, 2012,
483(7389): 336-340.
[72] 吴荣波, 李丕龙. 液-液相分离与生物分子凝集体[J]. 科学通报, 2019, 64(22): 2285-2291.
[73] ZABOROWSKA J, EGLOFF S, MURPHY S. The pol II CTD: new twists in the tail [J]. Nature Structural & Molecular Biology, 2016, 23(9): 771-777.
[74] MEINHART A, KAMENSKI T, HOEPPNER S, et al. A structural perspective of CTD function [J]. Genes & Development, 2005,
19(12): 1401-1415.
[75] HSIN J P, MANLEY J L. The RNA polymerase II CTD coordinates transcription and RNA processing [J]. Genes & Development, 2012, 26(19): 2119-2137.
[76] LU F Y, PORTZ B, GILMOUR D S. The C-terminal domain of RNA polymerase II is a multivalent targeting sequence that supports drosophila development with only consensus heptads [J]. Molecular Cell, 2019, 73(6): 1232-1242.e4.
[77] SAWICKA A, VILLAMIL G, LIDSCHREIBER M, et al. Transcription activation depends on the length of the RNA polymerase II C-terminal domain [J]. The EMBO Journal, 2021, 40(9): e107015.
[78] BOEHNING M, DUGAST-DARZACQ C, RANKOVIC M, et al. RNA polymerase II clustering through carboxy-terminal domain
phase separation [J]. Nature Structural & Molecular Biology, 2018, 25(9): 833-840.
[79] CHEN X Z, WEI M, ZHENG M M, et al. Study of RNA polymerase II clustering inside live-cell nuclei using bayesian nanoscopy [J]. Acs Nano, 2016, 10(2): 2447-2454.
[80] CHO W K, SPILLE J H, HECHT M, et al. Mediator and RNA polymerase II clusters associate in transcription-dependent
condensates [J]. Science, 2018, 361(6400): 412-415.
[81] BOIJA A, KLEIN I A, SABARI B R, et al. Transcription factors activate genes through the phase-separation capacity of their
activation domains [J]. Cell, 2018, 175(7): 1842-1855.e16.
[82] SABARI B R, DALL'AGNESE A, BOIJA A, et al. Coactivator condensation at super-enhancers links phase separation and gene
control [J]. Science, 2018, 361(6400): eaar3958.
[83] VOJNOVIC I, WINKELMEIER J, ENDESFELDER U. Visualizing the inner life of microbes: practices of multi-color single-molecule
localization microscopy in microbiology [J]. Biochemical Society Transactions, 2019, 47(4): 1041-1065.
[84] CABRERA J E, JIN D J. Active transcription of rRNA operons is a driving force for the distribution of RNA polymerase in
bacteria: Effect of extrachromosomal copies of rrnB on the in vivo localization of RNA polymerase [J]. Journal of Bacteriology, 2006, 188(11): 4007-4014.
[85] JIN D J, MATA MARTIN C, SUN Z, et al. Nucleolus-like compartmentalization of the transcription machinery in fast-growing
bacterial cells [J]. Critical Reviews in Biochemistry and Molecular Biology, 2017, 52(1): 96-106.
[86] CABRERA J E, JIN D J. The distribution of RNA polymerase in Escherichia coli is dynamic and sensitive to environmental cues [J]. Molecular Microbiology, 2003, 50(5): 1493-1505.
[87] STRACY M, LESTERLIN C, GARZA DE LEON F, et al. Livecell superresolution microscopy reveals the organization of RNA
polymerase in the bacterial nucleoid [J]. Proceedings of the National Academy of Sciences of the United States of America, 2015,
112(32): E4390-E4399.
[88] WENG X, BOHRER C H, BETTRIDGE K, et al. Spatial organization of RNA polymerase and its relationship with transcription in Escherichia coli [J]. Proceedings of the National Academy of Sciences of the United States of America, 2019, 116(40): 20115-20123.
[89] LADOUCEEUR A M, PARMAR B S, BIEDZINSKI S, et al. Clusters of bacterial RNA polymerase are biomolecular condensates
that assemble through liquid-liquid phase separation [J]. Proceedings of the National Academy of Sciences of the United States of
America, 2020, 117(31): 18540-18549.
[90] BREMER H, DENNIS P P. Modulation of chemical composition and other parameters of the cell at different exponential growth rates [J]. EcoSal Plus, 2008, 3(1). DOI: 10.1128/ecosal.5.23.
[91] SHIN Y, BRANGWYNNE C P. Liquid phase condensation in cell physiology and disease [J]. Science, 2017, 357(6357): eaaf4382.
[92] 江海燕, 吴旻昊. 生物大分子相分离研究进展和发展建议[J]. 科学通报, 2020, 65(20): 2085-2093.
[93] SHRINIVAS K, SABARI B R, COFFEY E L, et al. Enhancer features that drive formation of transcriptional condensates [J].
Molecular Cell, 2019, 75(3): 549-561.e7.
[94] STRACY M, KAPANIDIS A N. Single-molecule and superresolution imaging of transcription in living bacteria [J]. Methods,
2017, 120: 103-114.