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J. Biol. Chem., Vol. 279, Issue 2, 1343-1350, January 9, 2004

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Enzymatic Mechanism of RNA Translocation in Double-stranded RNA Bacteriophages^*

Jií Lísal, Denis E. Kainov, Dennis H. Bamford, George J. Thomas, Jr., and Roman Tuma¶

From the Department of Biosciences and Institute of Biotechnology, University of Helsinki, 00014 Finland and the Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri, Kansas City, Missouri 64110

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.

Received for publication, August 28, 2003 , and in revised form, September 26, 2003.

^* This work was supported by the Academy of Finland, "Finnish Centre of Excellence Program 2000-2005," Grants 172623 (to R. T.) and 1202855 (to D. H. B) and United States National Institutes of Health Grant GM50776 (to G. J. T.).

^¶ To whom correspondence should be addressed: Viikki Biocenter, P. O. Box 65, Viikinkaari 1, FIN-00014, University of Helsinki, Finland. Tel.: 358-9-191-59577; Fax: 358-9-191-59930; E-mail: roman.tuma{at}helsinki.fi.

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