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Single-molecule analysis of structure and function of human transcription complexes using DNA origami nano-robots.

Primary Supervisor: Dr Andrey Revyakin, Department of Molecular and Cell Biology

Secondary Supervisor: Professor Daniel Panne

PhD project title: Single-molecule analysis of structure and function of human transcription complexes using DNA origami nano-robots

University of Registration: University of Leicester

Project outline:

Project outline describing the scientific rationale of the project (max 4,000 characters incl. spaces and returns). There is no need to be too detailed about individual projects – general background, objectives and methods should suffice. One or two key references for potential applicants would be useful.

DNA origami is a rapidly evolving area of nanotechnology in which self-assembly properties of the DNA double helix are harnessed to build three-dimensional structures on the dimensional scale of ~50 nm. Intriguingly, this scale also happens to be the characteristic size of macromolecular complexes involved in the process of gene expression. In this multidisciplinary project, you will combine DNA origami technology with single-molecule spectroscopy, biochemistry, and CRISPR gene-editing to study the assembly and the dynamic structure of macromolecular protein complexes involved in human mRNA transcription.

Transcription of mRNA begins with the binding of RNA polymerase II (RNAPII), assisted by six General Transcription Factors (GTFs) and activators, to the promoter of the respective gene. The complex comprised of the promoter, activators, GTFs, and RNAPII is called the ‘pre-initiation complex’ (PIC), a macromolecular assembly about 30 nm in size. Once the PIC is formed, RNAPII unwinds the promoter, initiates mRNA synthesis, and escapes the promoter while transcribing mRNA. A central question of molecular biology is how activators, RNAP II and GTFs ‘know’ at which promoters, how frequently, and for how long to assemble into a PIC.

The molecular structures of RNAP II, GTFs, activators and, most recently, whole PICs have been studied using X-ray crystallography, Cryo-electron microscopy (CryoEM) and biochemical techniques. However, it still remains unclear in what order the components of the PIC assemble, what rate-limiting steps must to be overcome, and what happens to the PIC once RNAPII begins transcription. To tackle this question, the Revyakin lab has developed a single-molecule imaging technique to visualize human transcription initiation in real-time. In this method, single promoter DNA molecules are captured in a solid-state fluidic chamber, the locations of the molecules are mapped to within a few nanometres, and fluorescently-tagged human RNAPII and GTFs are allowed to initiate transcription on the mapped DNA (single-molecule transcription, SMT). During SMT, assembly of PICs is visualized in real-time, and the interactions between proteins and DNA are inferred from the appearance of fluorescence spots co-localizing with each other. Most recently, the Revyakin lab used this approach to capture, at 1-second resolution, the dynamic entry of individual GTF and RNAPII molecules into single PICs, followed by transcription initiation, escape, elongation, and re-initiation by single RNAP II molecules.

In this PhD project you will build a space-time movie of assembly of human RNAPII preinitiation complexes on promoters. Specifically, you will use the single-molecule technique by the Revyakin lab to capture not only the time of entry of GTFs and RNAPII into PICs, but also map the positions of these protein molecules with respect to each other and the promoter. The mapping of the protein molecules will be enabled by the use of novel ‘high-bar’ DNA origami nanorobots. In these ‘high-bars’, single promoter DNA fragments are stretched between two nano-pillars, effectively pinning down the promoter in three dimensions, restricting its Brownian motion, and allowing localization of GTFs and RNAP II molecules with the precision and accuracy better than the size of individual PICs and the size of the promoter (~10 nm). Your real-time single-molecule structural data will be complemented by studies obtained by high-resolution Cryo-EM structures of activator-PIC complexes (a study underway at the group of Prof. Panne at LISCB, the co-supervisor in this project).

This project will provide a fundamentally new view of the RNAPII transcription process, and will also prepare the ground for deployment of origami nanorobots to probe transcriptional dynamics inside living cells. The project is comprised of three specific objectives:

In Objective 1, you will reconstitute in vitro transcription by human RNAPII, using DNA origami ‘high-bars’ as a template. Purified human RNAPII GTFs, and high-bar DNA origamis are already available at the Revyakin lab.

In Objective 2, you will purify, fluorescently label, and assay human RNAPII and two key GTFs -- TFIID (responsible for promoter binding) and TFIIH (responsible for promoter melting). The protein complexes will need to be prepared from a human cell line containing CRISPR-edited genes encoding for key tagged subunits of the respective factors. Pilot studies using tagged TFIID and TFIIH isolated from CRISPR-edited cells have already been completed by the Revyakin lab.

In Objective 3, you will use single-molecule microscopy to reconstitute ‘high-bar’ transcription using labelled RNAPII, and the labelled transcription factors TFIIH and TFIID. You will then optimize conditions (type of fluorescent label, irradiation, exposure, sampling rate, immobilization conditions, imaging buffers) for mapping of positions of single factor molecules to within 10 nm resolution, which is sufficient to resolve the positions of the three proteins with respect to each other and the transcription start site.

BBSRC Strategic Research Priority: Understanding the Rules of Life: Sturctural Biology

Techniques that will be undertaken during the project:

  • Real-time single-molecule biochemistry and super-resolution microscopy
  • DNA origamis: design (CADNANO), assembly, and purification (ultracentrifugation)
  • Protein expression and purification
  • Radioactivity-based biochemical assays
  • Bio-conjugation
  • Digital signal processing and data analysis (Matlab, Python)
  • CRISPR gene editing
  • Mechanical engineering, rapid prototyping, end-milling.
  • Molecular cloning

Contact: Dr Andrey Revyakin, University of Leicester