Leicester Institute of Structural and Chemical Biology

Andrey Revyakin

Single molecule dynamics of gene expression

Research summary

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We are a multi-disciplinary group interested in the intricate molecular mechanism of transcription regulation. Specifically, we use multi-colour super-resolution localisation microscopy, DNA nanotechnology and CRISPR gene-editing to visualise, in real-time, the dynamics of assembly of single molecules of transcription factors and RNA polymerases on promoters during transcription initiation.

There are three main projects ongoing at the lab. The first project tackles the single-molecule mechanism of transcription initiation by RNA polymerase II (pol 2) -- the enzyme that transcribes all protein-encoding genes in mammalian cells, and serves as the main control hub for all cell-state decisions. Our aim is to gain a real-time picture of transcription factor-dependent promoter recruitment of pol2 and promoter escape by pol 2. We have recently developed a single-molecule technology to image the assembly of the pol 2 transcription machinery on promoters in real-time, in an in vitro system reconstituted from labelled components. Our next goal is to visualise the real-time assembly of transcription initiation complexes on endogenous promoters in living human cells.

The second project tackles the molecular mechanism of transcription initiation using the human mitochondrial RNA polymerase (mtRNAP) as model system. Like nuclear RNAPs, mtRNAP cannot find promoters in the mitochondrial genome on its own, and requires general transcription factors to initiate transcription. To visualise the dynamics of mtRNAP transcription initiation at single-molecule resolution, we use a reconstituted system in which all components of the mtRNAP machinery are fluorescently labelled, and promoter assembly and RNA production are probed in real-time by super-resolution microscopy and co-localisation.

The third project deals with pushing the limits of time- and spatial resolution achievable by single-molecule imaging, and by optical systems in general. Despite the cutting-edge aspect of single-molecule fluorescence detection, the technique is still limited by the diffraction of light. The diffraction limit largely prevents tracking single molecules at high concentrations (above >10 nM), a situation most typical in live cells. We are developing a universal, cost-effective approach to manipulate light beyond the diffraction limit that can be used both in vitro and in vivo. The method is based on self-assembled devices that we call DNA origami-based nanoantennas (or DONNAs). The specific applications of DONNAs that we envision are fast, accurate, affordable DNA sequencing (critical for personalised medicine), and visualisation of processes in live human cells at single-molecule resolution (critical for more efficient drug discovery).

Key publications

Group members

Rory Cunnison.

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