Single-Molecule Narrow-Field Microscopy of Protein-DNA Binding Dynamics in Glucose Signal Transduction of Live Yeast Cells.
Single-molecule narrow-field microscopy is a versatile tool to investigate a diverse range of protein dynamics in live cells and has been extensively used in bacteria. Here, we describe how these methods can be extended to larger eukaryotic, yeast cells, which contain subcellular compartments. We describe how to obtain single-molecule microscopy data but also how to analyze these data to track and obtain the stoichiometry of molecular complexes diffusing in the cell. We chose glucose mediated signal transduction of live yeast cells as the system to demonstrate these single-molecule techniques as transcriptional regulation is fundamentally a single-molecule problem-a single repressor protein binding a single binding site in the genome can dramatically alter behavior at the whole cell and population level.
Biological Physical Sciences Institute (BPSI), University of York, Heslington, York, YO10 5DD, UK. adam.wollman@york.ac.uk.
Adam J M
AJ
Wollman
Biological Physical Sciences Institute (BPSI), University of York, Heslington, York, YO10 5DD, UK.
Mark C
MC
Leake
D001665:Binding Sites; D053898:Biosynthetic Pathways; D004271:DNA, Fungal / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D005947:Glucose / Q000378:metabolism; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D000072760:Single Molecule Imaging / Q000379:methods*
D004271:DNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D005947:Glucose
Signal transduction; Single-molecule biophysics; Yeast