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You are here: Home Administration Chemistry & Biochemistry Department Events 2018 March Biochemistry Seminar: Alexandros Pertsinidis, "Single-molecule studies of transcription mechanisms"

Biochemistry Seminar: Alexandros Pertsinidis, "Single-molecule studies of transcription mechanisms"

Alexandros Pertsinidis, Associate Member, Memorial Sloan Kettering Cancer Center, will give a talk on "Single-molecule studies of transcription mechanisms."
When May 23, 2018
from 12:00 PM to 01:00 PM
Where CUNY ASRC 5th Floor Data Visualization Room
Contact Name
Contact Phone 212-650-8803
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ABSTRACT

Transcription regulation employs 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 the intricate transcriptional machinery works. In addition, a following the activity of single molecules at specific gene loci inside live cells remains very challenging, the organization and dynamics of transcription in the nucleus remain largely uncharacterized.

We have developed novel single-molecule 3D imaging techniques that can visualize the dynamics of large macro-molecular machines in action and we have observed, for the first time, how RNA Polymerase II molecules progress through the transcription cycle at a single gene in live human cells, in real-time. The new insights are enabled by two new technologies: (i) modulation  interferometry, a 3D single-molecule super-resolution imaging approach that achieves dynamic real-time tracking with <2 nanometer axial localization precision, well below the few-nanometer size of individual protein components of large transcription complexes; (ii) target-locking nanoscopy, an ultrasensitive system that enables single-molecule detection in addressable subdiffraction volumes, at high background concentrations within crowded intracellular environments.

With the new capabilities of modulation inteferometry we have visualized the movement of a multi-subunit RNA Polymerase through the complete transcription cycle, dissected the kinetics and conformational changes of the initiation-elongation transition and determined the fate of an initiation factor during promoter escape. Our results validate modulation interferometry as one of few structural biology tools that presently have the combined spatial-temporal resolution and molecular specificity required to capture the movement, conformational changes and sub-unit association-dissociation kinetics of complex macromolecular machines, like the transcription apparatus, in action. Our newly-developed target-locking nanoscopy system enables us to image, track and count single RNA Polymerase II molecules, in real-time inside live human cells. We discovered ~10 Pol II molecules accumulating during active transcription of a tagged mini-gene. Kinetic analysis reveals that mini-gene transcription does not involve transient Pol II clustering at pre-initiation, persistence of accumulated Pol IIs in the absence of transcription or extensive Pol II recycling-related spatial compartmentalization. Rather, single Pol II molecules are stochastically recruited from the nucleoplasm, enter into productive elongation and are predominantly released rather than recycled upon termination. Our results demonstrate that complex biological mechanisms can be probed by real-time single-molecule detection in addressable 3D loci inside live cells and establish a quantitative framework for elucidating Pol II dynamics at single genes in vivo.

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