title: Binding of two PriA-PriB complexes to the primosome assembly site initiates primosome formation.
abstract: A direct quantitative analysis of the initial steps in primosome assembly, involving PriA and PriB proteins and the minimal primosome assembly site (PAS) of phage ϕX174, has been performed using fluorescence intensity, fluorescence anisotropy titration, and fluorescence resonance energy transfer techniques. We show that two PriA molecules bind to the PAS at both strong and weak binding sites on the DNA, respectively, without detectable cooperative interactions. Binding of the PriB dimer to the PriA-PAS complex dramatically increases PriA's affinity for the strong site, but only slightly affects its affinity for the weak site. Associations with the strong and weak sites are driven by apparent entropy changes, with binding to the strong site accompanied by a large unfavorable enthalpy change. The PriA-PriB complex, formed independently of the DNA, is able to directly recognize the PAS without the preceding the binding of PriA to the PAS. Thus, the high-affinity state of PriA for PAS is generated through PriA-PriB interactions. The effect of PriB is specific for PriA-PAS association, but not for PriA-double-stranded DNA or PriA-single-stranded DNA interactions. Only complexes containing two PriA molecules can generate a profound change in the PAS structure in the presence of ATP. The obtained results provide a quantitative framework for the elucidation of further steps in primosome assembly and for quantitative analyses of other molecular machines of cellular metabolism.
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch at Galveston, Galveston, TX 77555-1053, USA.
authors.forename: Michal R
authors.initials: MR
authors.lastname: Szymanski
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Jezewska
authors.forename: Wlodzimierz
authors.initials: W
authors.lastname: Bujalowski
mesh_terms: D004265:DNA Helicases / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D008954:Models, Biological; D046912:Multiprotein Complexes / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D004279:DNA, Viral; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D046912:Multiprotein Complexes; C086004:PriB protein, E coli; C506743:priA protein, E coli; D004265:DNA Helicases