The life of a cell is governed by the regulated transcription of genomic information from DNA to RNA by the enzyme RNA polymerase. Transcriptional control occurs through the actions of hundreds of proteins in the cell nucleus whose identities and functions have been elucidated from decades of genetic and biochemical studies. However, little is known about the timescales under which transcription-related proteins search for and target chromatin sites in living cells, and how such dynamic associations are influenced by modifications of chromatin architecture. This proposal aims to address these questions by applying super-resolution fluorescence microscopy to directly visualize the diffusive movements of major transcription initiation proteins at single- molecule resolution in budding yeast, under normal and altered states of chromatin architecture. Specifically, we will test the hypothesis that chromatin association of transcription initiation proteins occurs with rapid kinetics in live cells, and that chromatin remodeling and modification has a role in regulating transcription protein dynamics. We will use live-cell single-molecule imaging to monitor the diffusive behavior of transcription initiation proteins at high spatio-temporal resolution in the yeast nucleus. This `in vivo biochemistry' approach differs from and is complementary to ChIP-Seq techniques that map steady-state occupancies genome-wide but provide little information on binding dynamics. We will engineer and functionally validate DNA constructs encoding components representative of the general transcription factors and major sequence-specific DNA binding transcription factors fused to a self-labeling protein tag (HaloTag) that can react covalently with a cell-permeable organic fluorophore (Janelia Fluor). Live-cell imaging of fluorescently labeled transcription factors at single- molecule resolution measures diffusion coefficients, distinguishes between chromatin-bound and chromatin-free populations, and estimates residence times for the bound population. Further, we will use conditional depletion of 6 major chromatin remodelers and histone modifiers to reveal changes in the mobilities of transcription initiation proteins and inform which among several diffusive parameters are subject to chromatin controls. This combination of conditional mutant genetics and live-cell single-molecule imaging may transform understanding of the kinetic mechanisms for transcription initiation and offer a new approach to other areas of yeast nuclear and chromosome biology, including studies of DNA replication, repair, and recombination.
The life of a cell is governed by the regulated transcription of genomic information from DNA to RNA by the enzyme RNA polymerase. Transcriptional control occurs through the actions of hundreds of proteins in the cell nucleus whose identities and functions have been elucidated from decades of genetic and biochemical studies. However, little is known about the timescales under which transcription-related proteins search for and target chromosomal sites in living cells, and how such dynamic associations are influenced by modifications of chromosome architecture. This proposal aims to apply super-resolution fluorescence microscopy to address these questions by directly visualizing the mobilities of major transcription proteins at single-molecule resolution, in living cells of the simple model organism, baker's yeast, under normal and altered chromosome states.
