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


Project Description

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 (representative video is available at https://www.youtube.com/watch?v=nqxpNgzrKiM).

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.

This is an extremely challenging, demanding project which will require full commitment and, most likely, will involve working beyond normal 9-5 hours.

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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
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Eligibility: UK/EU applicants only.

Entry requirements: Students with physics, biophysics, biochemistry, chemical physics, physical chemistry, and bioinformatics background are particularly encouraged to apply.

Applicants are required to hold/or expect to obtain a UK Bachelor Degree 2:1 or better in a relevant subject.

The University of Leicester English language requirements apply where applicable: https://le.ac.uk/study/research-degrees/entry-reqs/eng-lang-reqs/ielts-65

How to apply:
Please refer carefully to the application guidance and apply using the online application link at https://le.ac.uk/study/research-degrees/funded-opportunities/bbsrc-mibtp

Project / Funding Enquiries:
Application enquiries to
Informal inquiries: ar371_AT_le.ac.uk (replace AT and remove underscores)
Closing date for applications: Sunday 12th January 2020

Funding Notes

The project provides a monthly subsistence stipend for the student, and 5K per year in consumables, for 4 years.

References

Revyakin, A.*, Zhang, Z.*, Coleman, R.A., Li, Y., Lucas, J.K., Inouye C., Chu, S., Tjian, R. (2012). Visualization of transcription by human RNA Polymerase II at the single-molecule level. Genes Dev. 26 (15);

Ortega E, Rengachari S, Ibrahim Z, Hoghoughi N, Gaucher J, Holehouse AS, Khochbin S, Panne D (2018). Transcription factor dimerization activates the p300 acetyltransferase, Nature. 562(7728):538-544.

Zhang Z, English B, Grimm J, Kazane S, Hu W, Tsai A, Inouye C, You C, Piehler J, Schultz PG, Lavis L, Revyakin A, Tjian R (2016) Rapid Dynamics of General Transcription Factor TFIIB Binding During Preinitiation Complex Assembly Revealed by Single-Molecule Analysis. Genes Dev; 30(18):2106-118;

Zhang Z.,* Revyakin A.*, Grimm J.B., Lavis L.D., Tjian R. (2014) Single-molecule tracking of the transcription cycle by sub-second RNA detection. eLife:3:e01775;

Revyakin, A., Liu, C.-Y., Ebright, R.H., and Strick, T.R. (2006). Abortive initiation and promoter escape by RNA polymerase involves DNA scrunching. Science 314: 1139-43;

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