title: Single molecule tracking of Ace1p in Saccharomyces cerevisiae defines a characteristic residence time for non-specific interactions of transcription factors with chromatin.
abstract: In vivo single molecule tracking has recently developed into a powerful technique for measuring and understanding the transient interactions of transcription factors (TF) with their chromatin response elements. However, this method still lacks a solid foundation for distinguishing between specific and non-specific interactions. To address this issue, we took advantage of the power of molecular genetics of yeast. Yeast TF Ace1p has only five specific sites in the genome and thus serves as a benchmark to distinguish specific from non-specific binding. Here, we show that the estimated residence time of the short-residence molecules is essentially the same for Hht1p, Ace1p and Hsf1p, equaling 0.12-0.32 s. These three DNA-binding proteins are very different in their structure, function and intracellular concentration. This suggests that (i) short-residence molecules are bound to DNA non-specifically, and (ii) that non-specific binding shares common characteristics between vastly different DNA-bound proteins and thus may have a common underlying mechanism. We develop new and robust procedure for evaluation of adverse effects of labeling, and new quantitative analysis procedures that significantly improve residence time measurements by accounting for fluorophore blinking. Our results provide a framework for the reliable performance and analysis of single molecule TF experiments in yeast.
authors: CCR/LRBGE Optical Microscopy Core, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors: David A
authors: DA
authors: Ball
authors: CCR/LRBGE Optical Microscopy Core, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors: Gunjan D
authors: GD
authors: Mehta
authors: CCR/LRBGE Optical Microscopy Core, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors: Ronit
authors: R
authors: Salomon-Kent
authors: Istituto Scientifico Ospedale San Raffaele, Centro di Imaging Sperimentale e Università Vita-Salute San Raffaele, Milan 20132, Italy.
authors: Davide
authors: D
authors: Mazza
authors: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523, USA.
authors: Tatsuya
authors: T
authors: Morisaki
authors: Institut Pasteur, Computation Imaging and Modeling Unit, CNRS, URA 2582, Paris 75015, France.
authors: Florian
authors: 0000-0002-9622-4396
authors: F
authors: Mueller
authors: Institute for Soft Matter and Functional Materials, Helmholtz Center Berlin, Berlin 12489, Germany.
authors: James G
authors: JG
authors: McNally
authors: CCR/LRBGE Optical Microscopy Core, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA karpovat@mail.nih.gov.
authors: Tatiana S
authors: TS
authors: Karpova
mesh_terms: D002843:Chromatin / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006657:Histones / Q000235:genetics / Q000378:metabolism; D008668:Metallothionein / Q000235:genetics / Q000378:metabolism; D057054:Molecular Imaging / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014157:Transcription Factors / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*
chemical_list: C500769:CUP1-1 protein, S cerevisiae; C060256:CUP2 protein, S cerevisiae; D002843:Chromatin; D004268:DNA-Binding Proteins; C067168:HSF1 protein, S cerevisiae; D006360:Heat-Shock Proteins; D006657:Histones; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D008668:Metallothionein