A Single-Molecule Surface-Based Platform for Detecting the Assembly and Function of the Human RNA Polymerase II Transcription Machinery

Single-molecule detection and manipulation is a powerful tool for unraveling dynamic biological processes. Unfortunately, success in such experiments is often challenged by tethering the bio-molecule(s) of interest to a biocompatible surface. Here we describe a robust surface passivation method by d...

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Bibliographic Details
Published in:Structure (London) 2020-08, Vol.28 (12), p.1337-1343.e4
Main Authors: Park, Sang Ryul, Hauver, Jesse, Zhang, Yunxiang, Revyakin, Andrey, Coleman, Robert A., Tjian, Robert, Chu, Steven, Pertsinidis, Alexandros
Format: Article
Language:English
Online Access:Get full text
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Summary:Single-molecule detection and manipulation is a powerful tool for unraveling dynamic biological processes. Unfortunately, success in such experiments is often challenged by tethering the bio-molecule(s) of interest to a biocompatible surface. Here we describe a robust surface passivation method by dense polymer-brush grafting, based on optimized polyethylene-glycol (PEG) deposition conditions, exactly at the lower critical point of an aqueous biphasic PEG-salt system. The increased biocompatibility achieved, compared to PEG deposition in sub-optimal conditions away from the critical point, allowed us to successfully detect the assembly and function of a large macro-molecular machine, a fluorescent-labeled multi-subunit, human RNA Polymerase II Transcription Pre-Initiation Complex, on single, promoter-containing, surface-immobilized DNA molecules. This platform will enable probing the complex biochemistry and dynamics of large, multi-subunit macromolecular assemblies, such as during the initiation of human RNA Pol II transcription, at the single-molecule level. Park et al. develop optimized surface preparation procedures for single-molecule experiments. The increased biocompatibility achieved enables visualizing the assembly and function of a large, multi-component molecular machinery, the human RNA Polymerase II transcription pre-initiation complex, on a natural promoter, at the single-molecule level.
ISSN:0969-2126
1878-4186
DOI:10.1016/j.str.2020.07.009