Transcription Factor Clustering in Live Yeast Cells

Abstract

Genes are regulated by a family of proteins called transcription factors which operate by binding to specific sequences of DNA. These sequences are short and occur frequently in the genome and it is unclear how transcription factors find their correct sites. We have investigated two zinc-finger transcription factors, Mig11 and Msn2, which are both glucose dependant repressors of genes. These proteins have been labelled with the green fluorescent protein (GFP) by chromosomal integration and another protein Nrd1 has been labelled with the mcherry fluorescent protein as a marker for the nucleus. Using single-molecule fluorescence microscopy2 in different extra-cellular glucose conditions, we are able to observe individual molecules of these proteins at different stages: bound to DNA in the nucleus, translocating across the nuclear membrane and diffusing in the cytoplasm. We show that these proteins operate in clusters through all these stages, in the nucleus and cytoplasm. Forming clusters may facilitate transcription factors finding their correct sites3 on the genome and provide possible evidence for transcription factories.1. Wollman, A. & Leake, M. C. FD2015-Single Molecule Microscopy: Millisecond single-molecule localization microscopy combined with convolution analysis and automated image segmentation to determine protein concentrations in complexly structured, functional cells, one cell at a time. Faraday Discuss. (2015). http://dx.doi.org/10.1039/C5FD00077G.2. Wollman, A. J. M., Miller, H., Zhou, Z. & Leake, M. C. Probing DNA interactions with proteins using a single-molecule toolbox: inside the cell, in a test tube and in a computer. Biochem. Soc. Trans. 43, 139-145 (2015).3. Schmidt, H. G., Sewitz, S., Andrews, S. S. & Lipkow, K. An Integrated Model of Transcription Factor Diffusion Shows the Importance of Intersegmental Transfer and Quaternary Protein Structure for Target Site Finding. 9, (2014).