Enzymatic mechanism of RNA translocation in double-stranded RNA bacteriophages.
Many complex viruses acquire their genome by active packaging into a viral precursor particle called a procapsid. Packaging is performed by a viral portal complex, which couples ATP hydrolysis to translocation of nucleic acid into the procapsid. The packaging process has been studied for a variety of viruses, but the mechanism of the associated ATPase remains elusive. In this study, the mechanism of RNA translocation in double-stranded RNA bacteriophages is characterized using rapid kinetic analyses. The portal complex of bacteriophage 8 is a hexamer of protein P4, which exhibits nucleotide triphosphatase activity. The kinetics of ATP binding reveals a two-step process: an initial, fast, second-order association, followed by a slower, first-order phase. The slower phase exhibits a high activation energy and has been assigned to a conformational change. ATP binding becomes cooperative in the presence of RNA. Steady-state kinetics of ATP hydrolysis, which proceeds only in the presence of RNA, also exhibits cooperativity. On the other hand, ADP release is fast and RNA-independent. The steady-state rate of hydrolysis increases with the length of the RNA substrate indicating processive translocation. Raman spectroscopy reveals that RNA binds to P4 via the phosphate backbone. The ATP-induced conformational change affects the backbone of the bound RNA but leaves the protein secondary structure unchanged. This is consistent with a model in which cooperativity is induced by an RNA link between subunits of the hexamers and translocation is effected by an axial movement of the subunits relative to one another upon ATP binding.
Lísal
Jíri
J
Department of Biosciences and Institute of Biotechnology, University of Helsinki, 00014 Finland.
Kainov
Denis E
DE
Bamford
Dennis H
DH
Thomas
George J
GJ
Tuma
Roman
R
D000244:Adenosine Diphosphate / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D001435:Bacteriophages / Q000737:chemistry / Q000378:metabolism*; D001692:Biological Transport; D002384:Catalysis; D004305:Dose-Response Relationship, Drug; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D009696:Nucleic Acids / Q000737:chemistry; D009711:Nucleotides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D012313:RNA / Q000737:chemistry*; D012330:RNA, Double-Stranded / Q000737:chemistry*; D013059:Spectrum Analysis, Raman; D013696:Temperature
D009696:Nucleic Acids; D009711:Nucleotides; D012330:RNA, Double-Stranded; D000244:Adenosine Diphosphate; D012313:RNA; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases