Cooperative mechanism of RNA packaging motor.
P4 is a hexameric ATPase that serves as the RNA packaging motor in double-stranded RNA bacteriophages from the Cystoviridae family. P4 shares sequence and structural similarities with hexameric helicases. A structure-based mechanism for mechano-chemical coupling has recently been proposed for P4 from bacteriophage phi12. However, coordination of ATP hydrolysis among the subunits and coupling with RNA translocation remains elusive. Here we present detailed kinetic study of nucleotide binding, hydrolysis, and product release by phi12 P4 in the presence of different RNA and DNA substrates. Whereas binding affinities for ATP and ADP are not affected by RNA binding, the hydrolysis step is accelerated and the apparent cooperativity is increased. No nucleotide binding cooperativity is observed. We propose a stochastic-sequential cooperativity model to describe the coordination of ATP hydrolysis within the hexamer. In this model the apparent cooperativity is a result of hydrolysis stimulation by ATP and RNA binding to neighboring subunits rather than cooperative nucleotide binding. The translocation step appears coupled to hydrolysis, which is coordinated among three neighboring subunits. Simultaneous interaction of neighboring subunits with RNA makes the otherwise random hydrolysis sequential and processive.
Lísal
Jirí
J
Department of Biological and Environmental Sciences and Institute of Biotechnology, University of Helsinki, Finland.
Tuma
Roman
R
D000244:Adenosine Diphosphate / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry; D001435:Bacteriophages / Q000737:chemistry; D001667:Binding, Competitive; D001692:Biological Transport; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D046911:Macromolecular Substances / Q000737:chemistry; D008954:Models, Biological; D009711:Nucleotides / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D011485:Protein Binding; D012313:RNA / Q000737:chemistry; D012317:RNA Phages / Q000737:chemistry*; D012330:RNA, Double-Stranded / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics; D013269:Stochastic Processes
D046911:Macromolecular Substances; D009711:Nucleotides; D010710:Phosphates; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D000244:Adenosine Diphosphate; D012313:RNA; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases