Single-molecule analysis of the full transcription cycle of the human mitochondrial transcription machinery

Lead Research Organisation: University of Leicester
Department Name: Molecular and Cell Biology

Abstract

Gene regulation is at the heart of all cell-state decisions. The commitment of a cell to turn on a gene is made primarily at the step of recruitment of RNA polymerase (RNAP) to the gene's promoter, regulated by numerous transcription factors (TFs). It still remains unclear how the signal from multiple TFs is integrated in space and time to 'switch' transcription on. To complicate matters further, each promoter is represented in the cell by only 1-2 molecules, which makes gene expression susceptible to molecular fluctuations at promoters. Direct single-molecule visualization of TF-promoter interactions is the most direct approach to trace how from a chaos of stochastic molecular interactions emerges a seemingly deterministic pattern of gene expression, cell-state decisions, and differentiation.
The molecular machinery responsible for switching on promoters in the nuclei of human cells is very complex: at least 50 polypeptides are required to melt the promoter in a reconstituted assay, and even more are required for regulation. In this project, we will use single-molecule super-resolution microscopy to tackle a much simpler human transcription system - the human mitochondrial transcription machinery. The system is comprised of only three components - mitochondrial RNA polymerase (mtRNAP) and two TFs - and yet it recapitulates the main events of transcription initiation by the nuclear RNA polymerases (i.e. assembly of the preinitiation complex, promoter bending, promoter melting, escape, and elongation). Thus, Emily will introduce fluorescent tags into mtRNAP and the two mtTFs, and visualize, in real-time the assembly disassembly of their preinitiation complex during the full transcription cycle, which will provide mechanistic insights into regulation of gene expression, and the role of stochastic fluctuations in transcriptional outcomes.

Publications

10 25 50

Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M01116X/1 01/10/2015 30/09/2023
1645650 Studentship BB/M01116X/1 05/10/2015 30/09/2019 Emily Rose Teece
 
Description Work on this grant has led to the development of the LESTASCOPE, our custom-designed open-source microscope for single-molecule imaging, and the engineering of a complementary temperature-controlled reaction environment. These technologies together allow the real-time visualisation of single, individual proteins; up to four different proteins can be monitored simultaneously by attaching different fluorescent dyes to each.

The data that can be generated using the LESTASCOPE provide individual information about every single protein-protein, protein-DNA or protein-RNA interaction that is recorded during our real-time imagine experiments. This kind of data regarding the stochastic dynamics of individual components is not something that could be accessed via the traditional biochemical and structural methods of protein study.

Given the sheer quantity of data that can be generated during just one experiment, and the bespoke nature of the system and data analysis in use, our analyses are currently only at a very early stage. Even so, from these we have observed many of the existing understandings of the human mitochondrial transcription system, including that a transcription factor A (TFAM) is the first component of the system to bind to DNA. We have also confirmed that TFAM, transcription factor B2 (TFB2M) and the mitochondrial RNA polymerase (mtRNAP) are essential for successful transcription initiation in vitro.

In addition, we are able to measure the rate of transcription initiation, the rate of elongation (i.e. how long it takes to produce a given transcript), and the rate of promoter escape by mtRNAP (i.e. how long it takes for mtRNAP to break away from its assembled complex with TFAM and TFB2M and initiate transcription). This alone gives us a much greater understanding of how genes in the mitochondrial genome are "switched on".

Whilst our analyses and experiments are still very much ongoing, we have also observed some unexpected behaviours. One such behaviour is that mtRNAP can, in the presence of TFAM, bind to DNA both transiently (i.e. non-productively) and non-transiently (productively). We have also seen that TFAM and mtRNAP can bind to the DNA and form a non-transient complex without TFB2M, which would contradict theories that the three proteins form a complex in solution before binding to a promoter. Finally, we have observed for the first time that TFAM can slide along DNA in both directions while bound to mtRNAP until it settles at the promoter and fires transcription. While this behaviour of TFAM on DNA has been observed alone before, this is the first suggestion that it is actually a constituent part of the process of locating promoters and initiating transcription.

Further work will include deeper analysis of the data regarding TFAM and mtRNAP already collected, as well as fluorescently-tagging TFB2M and observing its behaviour as the alleged final binding component of the human mitochondrial transcription system.
Exploitation Route While we have here applied the LESTASCOPE and associated reaction environment technology to the study of human mitochondrial transcription, it has the potential to be widely applicable to the academic study of any biological system in which various components assemble on a single component, as long as the system can be reconstituted in vitro and component on which that assembly takes place can be tethered at one end to allow visualisation. Whilst we have begun to understand the full complexity of the dynamics of human mitochondrial transcription, there is also more to learn, in particular regarding TFB2M, the only core component yet to be visualised - it looks likely that this, along with eventually adapting the LESTASCOPE for live-cell imaging, will form the basis for future work by our laboratory. Outside academia, the LESTASCOPE technology could be used in the pharmaceutical sector to study drug interactions; in addition, there is the hope that the information ascertained regarding human mitochondrial transcription might one day become the basis for targeted therapies to treat mitochondrial diseases.
Sectors Chemicals,Healthcare,Pharmaceuticals and Medical Biotechnology