Transcription is a process essential to all known forms of life. To understand the mechanisms involved, we track individual proteins of interest in live cells by labelling them with fluorescent tags.
Current controversies around the exact mechanisms within the transcription cycle, arising from single-molecule in vitro studies, and transcription spatial organisation in vivo cannot be resolved by ensemble methods, nor fully addressed faithfully in vitro. Using photoactivated localisation microscopy (PALM) and other fluorescent sleights of hand, we are able to resolve single molecules in a live cell; by fitting a geometric profile to the individual spots we can determine its location with a precision of around 40 nm, tracking the molecule by recording its position over time. Molecular tracks are then processed to obtain diffusion properties, spatial distributions, copy numbers, and clustering properties that reveal new information about this fundamentally important biological process.
We have directly investigated the activity of polymerases, the populations of RNA polymerases in Escherichia coli cells involved in target search and in active transcription on the bacterial chromosome, and mobility and spatial distribution of transcription factors, such as the lac repressor, using PALM. We have also found via clustering analyses that dense clusters of transcribing RNAPs occur almost exclusively at the nucleoid periphery, leading to more extensive cluster-based analysis to discern the spatial organisation of key processes in the cell. We are exploring the roles of highly-conserved transcriptional elongation regulators, NusG and RfaH, via single-molecule imaging of fluorescent protein derivatives and double-labelling for single molecule FRET studies, to give further insight into the complex control gene expression and transcription-translation coupling.
Our in vivo projects aim to elucidate the details of transcription cycles inside living cells, resolve the structure, composition, and physical nature of large RNAP clusters, and test for functions of RNAP clusters in promoter search and transcription re-initiation, using super-resolution imaging, microbiology, proteomics/transcriptomics, single-molecule localisation and tracking, and modelling.
