Transcription Factor Target Search Strategy At The Single Molecule Level In Eukaryotic Cells

Transcription is the mechanism by which information encoded in genes is transmitted to an mRNA template. This process is initiated by the binding of a transcription factor (TF) to a gene promoter. TFs have to find their specific gene promoters (∼20bp) within a 3.2 billion bps genome in a fast and efficient manner. A key challenge is thus to understand by which mechanisms do TFs execute target search in a crowded nucleus? To address this question, we use a non-native doxycycline-deactivable Tet Repressor (TetR) DNA-binding domain integrated into a human cell line (U2OS). By transfecting cells with TetR tagged with the photo-convertible DENDRA2, we are able to visualize their dynamics in the nuclei of live cells using a sptPALM technique. Low UV irradiation causes stochastic conversion of DENDRA2 from the green to the red, with an event probability low enough to detect single molecules. The nuclear dynamics of TetR exhibits three different behaviours: (I)trajectories that cross the entire nucleus, screening it predominantly with a fast motion (16μm²/s); (II)quasi-immobile molecules(0,1μm²/s), and (III)an intermediate behaviour, with medium motion(1,4μm²/s) confined in subparts of the nucleus. When doxycycline is added, disabling TetR DNA-binding properties, there is a drastic decrease of quasi-immobile molecules and a 50% decrease of the medium-motion ones. This suggests that the quasi-immobile molecules are unspecifically bound on DNA. Their slow motion suggests a 1dimensional screening of DNA as seen in in vitro experiments. The medium motion population shows transiant DNA unspecific binding combined with 3dimentional jumps, allowing a local search. The fast population screens in a global way the nucleus. TFs seem to have a well established target search strategy by unspecifically binding on DNA and combining 1Dimentional and 3Dimentional motions, as suggested by previous target search theories.