title: Single-Molecule Real-Time 3D Imaging of the Transcription Cycle by Modulation Interferometry.
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 σ70initiation 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.
authors.affiliation: 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.
authors.forename: Guanshi
authors.initials: G
authors.lastname: Wang
authors.affiliation: 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.
authors.forename: Jesse
authors.initials: J
authors.lastname: Hauver
authors.affiliation: Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.
authors.forename: Zachary
authors.initials: Z
authors.lastname: Thomas
authors.affiliation: The Rockefeller University, New York, NY 10065, USA.
authors.forename: Seth A
authors.initials: SA
authors.lastname: Darst
authors.affiliation: Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address: pertsina@mskcc.org.
authors.forename: Alexandros
authors.initials: A
authors.lastname: Pertsinidis
mesh_terms: D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods; D007368:Interferometry / Q000379:methods*; D000072760:Single Molecule Imaging / Q000379:methods*; D014158:Transcription, Genetic
chemical_list: D012321:DNA-Directed RNA Polymerases
keywords: RNA polymerase; axial localization; holoenzyme; interferometry; nuclear pore complex; sigma factor; single-molecule; super-resolution imaging; transcription; transcription cycle