Mechanism of Transcription Initiation at an Activator-dependent Promoter Defined by Single-molecule Observation

Overview

Journal Cell

Publisher Cell Press

Specialty Cell Biology

Date 2012 Feb 21

PMID 22341441

Citations 89

Authors

Larry J Friedman

Jeff Gelles

Affiliations

Soon will be listed here.

Abstract

Understanding the pathway and kinetic mechanisms of transcription initiation is essential for quantitative understanding of gene regulation, but initiation is a multistep process, the features of which can be obscured in bulk analysis. We used a multiwavelength single-molecule fluorescence colocalization approach, CoSMoS, to define the initiation pathway at an activator-dependent bacterial σ(54) promoter that recapitulates characteristic features of eukaryotic promoters activated by enhancer binding proteins. The experiments kinetically characterize all major steps of the initiation process, revealing heretofore unknown features, including reversible formation of two closed complexes with greatly differing stabilities, multiple attempts for each successful formation of an open complex, and efficient release of σ(54) from the polymerase core at the start of transcript synthesis. Open complexes are committed to transcription, suggesting that regulation likely targets earlier steps in the mechanism. CoSMoS is a powerful, generally applicable method to elucidate the mechanisms of transcription and other multistep biochemical processes.

Citing Articles

Real-time capture of σ transcription initiation intermediates reveals mechanism of ATPase-driven activation by limited unfolding.

Mueller A, Molina N, Darst S bioRxiv. 2025; .

PMID: 39974980 PMC: 11839083. DOI: 10.1101/2025.02.07.637174.

Anti-correlation of LacI association and dissociation rates observed in living cells.

Kandavalli V, Zikrin S, Elf J, Jones D Nat Commun. 2025; 16(1):764.

PMID: 39824877 PMC: 11748676. DOI: 10.1038/s41467-025-56053-z.

Single-molecule imaging for investigating the transcriptional control.

Choi I, Baek I Mol Cells. 2025; 48(2):100179.

PMID: 39814141 PMC: 11847471. DOI: 10.1016/j.mocell.2025.100179.

Kinetochores grip microtubules with directionally asymmetric strength.

Larson J, Heitkamp N, Murray L, Popchock A, Biggins S, Asbury C J Cell Biol. 2024; 224(1).

PMID: 39485274 PMC: 11533501. DOI: 10.1083/jcb.202405176.

Regulatory properties of transcription factors with diverse mechanistic function.

Ali M, Guharajan S, Parisutham V, Brewster R PLoS Comput Biol. 2024; 20(6):e1012194.

PMID: 38857275 PMC: 11192337. DOI: 10.1371/journal.pcbi.1012194.

References

Merrick M . In a class of its own--the RNA polymerase sigma factor sigma 54 (sigma N). Mol Microbiol. 1993; 10(5):903-9. DOI: 10.1111/j.1365-2958.1993.tb00961.x. View

Morris L, Cannon W, Claverie-Martin F, Austin S, Buck M . DNA distortion and nucleation of local DNA unwinding within sigma-54 (sigma N) holoenzyme closed promoter complexes. J Biol Chem. 1994; 269(15):11563-71. View

Rippe K, Guthold M, von Hippel P, Bustamante C . Transcriptional activation via DNA-looping: visualization of intermediates in the activation pathway of E. coli RNA polymerase x sigma 54 holoenzyme by scanning force microscopy. J Mol Biol. 1997; 270(2):125-38. DOI: 10.1006/jmbi.1997.1079. View

Browning D, Busby S . The regulation of bacterial transcription initiation. Nat Rev Microbiol. 2004; 2(1):57-65. DOI: 10.1038/nrmicro787. View

Bondarenko V, Liu Y, Ninfa A, Studitsky V . Action of prokaryotic enhancer over a distance does not require continued presence of promoter-bound sigma54 subunit. Nucleic Acids Res. 2002; 30(3):636-42. PMC: 100299. DOI: 10.1093/nar/30.3.636. View

Reitzer L, Schneider B . Metabolic context and possible physiological themes of sigma(54)-dependent genes in Escherichia coli. Microbiol Mol Biol Rev. 2001; 65(3):422-44, table of contents. PMC: 99035. DOI: 10.1128/MMBR.65.3.422-444.2001. View

Burrows P, Joly N, Cannon W, Camara B, Rappas M, Zhang X . Coupling sigma factor conformation to RNA polymerase reorganisation for DNA melting. J Mol Biol. 2009; 387(2):306-19. PMC: 2688459. DOI: 10.1016/j.jmb.2009.01.052. View

Deighan P, Pukhrambam C, Nickels B, Hochschild A . Initial transcribed region sequences influence the composition and functional properties of the bacterial elongation complex. Genes Dev. 2011; 25(1):77-88. PMC: 3012938. DOI: 10.1101/gad.1991811. View

Ishihama A . Functional modulation of Escherichia coli RNA polymerase. Annu Rev Microbiol. 2000; 54:499-518. DOI: 10.1146/annurev.micro.54.1.499. View

10.

Saecker R, Record Jr M, deHaseth P . Mechanism of bacterial transcription initiation: RNA polymerase - promoter binding, isomerization to initiation-competent open complexes, and initiation of RNA synthesis. J Mol Biol. 2011; 412(5):754-71. PMC: 3440003. DOI: 10.1016/j.jmb.2011.01.018. View

11.

Cannon W, Gallegos M, Buck M . Isomerization of a binary sigma-promoter DNA complex by transcription activators. Nat Struct Biol. 2000; 7(7):594-601. DOI: 10.1038/76830. View

12.

Cannon W, Claverie-Martin F, Austin S, Buck M . Core RNA polymerase assists binding of the transcription factor sigma 54 to promoter DNA. Mol Microbiol. 1993; 8(2):287-98. DOI: 10.1111/j.1365-2958.1993.tb01573.x. View

13.

Kontur W, Saecker R, Capp M, Record Jr M . Late steps in the formation of E. coli RNA polymerase-lambda P R promoter open complexes: characterization of conformational changes by rapid [perturbant] upshift experiments. J Mol Biol. 2008; 376(4):1034-47. PMC: 2396949. DOI: 10.1016/j.jmb.2007.11.064. View

14.

Reitzer L, Bueno R, Cheng W, Abrams S, Rothstein D, Hunt T . Mutations that create new promoters suppress the sigma 54 dependence of glnA transcription in Escherichia coli. J Bacteriol. 1987; 169(9):4279-84. PMC: 213741. DOI: 10.1128/jb.169.9.4279-4284.1987. View

15.

Ninfa A, Reitzer L, MAGASANIK B . Initiation of transcription at the bacterial glnAp2 promoter by purified E. coli components is facilitated by enhancers. Cell. 1987; 50(7):1039-46. DOI: 10.1016/0092-8674(87)90170-x. View

16.

Murakami K, Darst S . Bacterial RNA polymerases: the wholo story. Curr Opin Struct Biol. 2003; 13(1):31-9. DOI: 10.1016/s0959-440x(02)00005-2. View

17.

Bernardo L, Johansson L, Solera D, Skarfstad E, Shingler V . The guanosine tetraphosphate (ppGpp) alarmone, DksA and promoter affinity for RNA polymerase in regulation of sigma-dependent transcription. Mol Microbiol. 2006; 60(3):749-64. DOI: 10.1111/j.1365-2958.2006.05129.x. View

18.

Szalewska-Palasz A, Johansson L, Bernardo L, Skarfstad E, Stec E, Brannstrom K . Properties of RNA polymerase bypass mutants: implications for the role of ppGpp and its co-factor DksA in controlling transcription dependent on sigma54. J Biol Chem. 2007; 282(25):18046-18056. DOI: 10.1074/jbc.M610181200. View

19.

Sclavi B, Zaychikov E, Rogozina A, Walther F, Buckle M, Heumann H . Real-time characterization of intermediates in the pathway to open complex formation by Escherichia coli RNA polymerase at the T7A1 promoter. Proc Natl Acad Sci U S A. 2005; 102(13):4706-11. PMC: 555702. DOI: 10.1073/pnas.0408218102. View

20.

Cases I, de Lorenzo V, Ouzounis C . Transcription regulation and environmental adaptation in bacteria. Trends Microbiol. 2003; 11(6):248-53. DOI: 10.1016/s0966-842x(03)00103-3. View