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Cell. 2016 Dec 15;167(7):1839-1852.e21. doi: 10.1016/j.cell.2016.11.032.

Single-Molecule Real-Time 3D Imaging of the Transcription Cycle by Modulation Interferometry.

Author information

1
Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; BCMB Graduate Program, Weill Cornell Medicine, Cornell University, New York, NY 10065, USA.
2
Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Tri-Institutional PhD Program in Chemical Biology, New York, NY 10065, USA; The Rockefeller University, New York, NY 10065, USA.
3
Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.
4
The Rockefeller University, New York, NY 10065, USA.
5
Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address: pertsina@mskcc.org.

Abstract

Many essential cellular processes, such as gene control, employ elaborate mechanisms involving the coordination of large, multi-component molecular assemblies. Few structural biology tools presently have the combined spatial-temporal resolution and molecular specificity required to capture the movement, conformational changes, and subunit association-dissociation kinetics, three fundamental elements of how such intricate molecular machines work. Here, we report a 3D single-molecule super-resolution imaging study using modulation interferometry and phase-sensitive detection that achieves <2 nm axial localization precision, well below the few-nanometer-sized individual protein components. To illustrate the capability of this technique in probing the dynamics of complex macromolecular machines, we visualize the movement of individual multi-subunit E. coli RNA polymerases through the complete transcription cycle, dissect the kinetics of the initiation-elongation transition, and determine the fate of σ70 initiation factors during promoter escape. Modulation interferometry sets the stage for single-molecule studies of several hitherto difficult-to-investigate multi-molecular transactions that underlie genome regulation.

KEYWORDS:

RNA polymerase; axial localization; holoenzyme; interferometry; nuclear pore complex; sigma factor; single-molecule; super-resolution imaging; transcription; transcription cycle

PMID:
27984731
PMCID:
PMC5444671
DOI:
10.1016/j.cell.2016.11.032
[Indexed for MEDLINE]
Free PMC Article

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