Quantitative study of synthetic Hox transcription factor-DNA interactions in live cells.
Transcription factor-DNA interactions are life sustaining and therefore the subject of intensive research. In spite of vast effort, quantitative in vivo studies of the molecular mechanisms underlying these fundamental interactions remain challenging. In the preceding paper, we designed synthetic Sex combs reduced (Scr) peptides and validated genetically their function as transcriptional regulators. Here we present a controllable system for quantitative studies of protein-DNA interactions in live cells that enables us to "titrate" the concentration of the synthetic Scr peptides in a single cell. Using methods with single-molecule sensitivity, advanced fluorescence imaging and fluorescence correlation spectroscopy (FCS), we were able to study the kinetics of Scr-DNA interactions in live salivary gland cells, where Scr is normally expressed during development. We discerned freely moving Scr molecules, characterized the specific and nonspecific Scr peptide-DNA interactions, and estimated their corresponding dissociation constants (K(d)) in vivo. Our results suggest that the synthetic Scr transcription factors find their specific target sites primarily by multiple association/dissociation events, the rapidity of which is largely owed to electrostatic interactions. Based on these new findings, we formulate a model mechanism and emulate the kinetics of Scr homeodomain-DNA interactions in live cells using numerical simulations.
Department of Clinical Neuroscience, Karolinska Institutet, 17176 Stockholm, Sweden.
Vladana
V
Vukojevic
Dimitrios K
DK
Papadopoulos
Lars
L
Terenius
Walter J
WJ
Gehring
Rudolf
R
Rigler
D000818:Animals; D004247:DNA / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000378:metabolism*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
D018398:Homeodomain Proteins; D004247:DNA