title: Single-molecule imaging of DNA curtains reveals mechanisms of KOPS sequence targeting by the DNA translocase FtsK.
abstract: FtsK is a hexameric DNA translocase that participates in the final stages of bacterial chromosome segregation. Here we investigate the DNA-binding and translocation activities of FtsK in real time by imaging fluorescently tagged proteins on nanofabricated curtains of DNA. We show that FtsK preferentially loads at 8-bp KOPS (FtsK Orienting Polar Sequences) sites and that loading is enhanced in the presence of ADP. We also demonstrate that FtsK locates KOPS through a mechanism that does not involve extensive 1D diffusion at the scale of our resolution. Upon addition of ATP, KOPS-bound FtsK translocates in the direction dictated by KOPS polarity, and once FtsK has begun translocating it does not rerecognize KOPS from either direction. However, FtsK can abruptly change directions while translocating along DNA independent of KOPS, suggesting that the ability to reorient on DNA does not arise from DNA sequence-specific effects. Taken together, our data support a model in which FtsK locates KOPS through random collisions, preferentially engages KOPS in the ADP-bound state, translocates in the direction dictated by the polar orientation of KOPS, and is incapable of recognizing KOPS while translocating along DNA.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA.
authors.forename: Ja Yil
authors.initials: JY
authors.lastname: Lee
authors.forename: Ilya J
authors.initials: IJ
authors.lastname: Finkelstein
authors.forename: Estelle
authors.initials: E
authors.lastname: Crozat
authors.forename: David J
authors.initials: DJ
authors.lastname: Sherratt
authors.forename: Eric C
authors.initials: EC
authors.lastname: Greene
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D009690:Nucleic Acid Conformation
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C096452:FtsK protein, E coli; D008565:Membrane Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate