title: DNA binding proteins explore multiple local configurations during docking via rapid rebinding.
abstract: Finding the target site and associating in a specific orientation are essential tasks for DNA-binding proteins. In order to make the target search process as efficient as possible, proteins should not only rapidly diffuse to the target site but also dynamically explore multiple local configurations before diffusing away. Protein flipping is an example of this second process that has been observed previously, but the underlying mechanism of flipping remains unclear. Here, we probed the mechanism of protein flipping at the single molecule level, using HIV-1 reverse transcriptase (RT) as a model system. In order to test the effects of long-range attractive forces on flipping efficiency, we varied the salt concentration and macromolecular crowding conditions. As expected, increased salt concentrations weaken the binding of RT to DNA while increased crowding strengthens the binding. Moreover, when we analyzed the flipping kinetics, i.e. the rate and probability of flipping, at each condition we found that flipping was more efficient when RT bound more strongly. Our data are consistent with a view that DNA bound proteins undergo multiple rapid re-binding events, or short hops, that allow the protein to explore other configurations without completely dissociating from the DNA.
authors.affiliation: Kavli Institute of Nanoscience, Department of Bionanoscience, TU Delft, 2629HZ, Delft, The Netherlands.
authors.forename: Mahipal
authors.initials: M
authors.lastname: Ganji
authors.affiliation: Kavli Institute of Nanoscience, Department of Bionanoscience, TU Delft, 2629HZ, Delft, The Netherlands.
authors.forename: Margreet
authors.initials: M
authors.lastname: Docter
authors.affiliation: Basic Research Laboratory, National Cancer Institute, Frederick, MD 21702, USA.
authors.forename: Stuart F J
authors.initials: SF
authors.lastname: Le Grice
authors.affiliation: Kavli Institute of Nanoscience, Department of Bionanoscience, TU Delft, 2629HZ, Delft, The Netherlands e.a.abbondanzieri@tudelft.nl.
authors.forename: Elio A
authors.initials: EA
authors.lastname: Abbondanzieri
mesh_terms: D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D017931:DNA Primers / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000378:metabolism; D007477:Ions; D007700:Kinetics; D046911:Macromolecular Substances / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000378:metabolism; D011485:Protein Binding
chemical_list: D017931:DNA Primers; D004268:DNA-Binding Proteins; D007477:Ions; D046911:Macromolecular Substances; D009711:Nucleotides; D004247:DNA; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase