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

Sang Ryul Park, Jesse Hauver, Yunxiang Zhang, Andrey Revyakin, Robert A. Coleman, Robert Tjian, Steven Chu, Alexandros Pertsinidis

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

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 biomolecule(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 with PEG deposition in sub-optimal conditions away from the critical point, allowed us to successfully detect the assembly and function of a large macromolecular 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.

Original languageEnglish (US)
Pages (from-to)1337-1343.e4
JournalStructure
Volume28
Issue number12
DOIs
StatePublished - Dec 1 2020
Externally publishedYes

Keywords

  • PEG
  • Pre-Initiation Complex
  • RNA Polymerase
  • general transcription factors
  • pol II
  • polyethylene glycol
  • polymer brushes
  • single-molecule assays
  • surface passivation
  • transcription

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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