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J. Biol. Chem., Vol. 283, Issue 18, 12227-12231, May 2, 2008

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Initial Location of the RNA-dependent RNA Polymerase in the Bacteriophage 6 Procapsid Determined by Cryo-electron Microscopy^*

Anindito Sen¹, J. Bernard Heymann², Naiqian Cheng, Jian Qiao, Leonard Mindich, and Alasdair C. Steven

From the Laboratory of Structural Biology Research, NIAMS, National Institutes of Health, Bethesda, Maryland 20892 and the Department of Microbiology, The Public Health Research Institute Center, New Jersey Medical School, University of Medicine & Dentistry of New Jersey, Newark, New Jersey 07103

The RNA-dependent RNA polymerases (RdRPs) of Cystoviridae bacteriophages, like those of eukaryotic viruses of the Reoviridae, function inside the inner capsid shell in both replication and transcription. In bacteriophage 6, this inner shell is first assembled as an icosahedral procapsid with recessed 5-fold vertices that subsequently undergoes major structural changes during maturation. The tripartite genome is packaged as single-stranded RNA molecules via channels on the 5-fold vertices, and transcripts probably exit the mature capsid by the same route. The RdRP (protein P2) is assembled within the procapsid, and it was thought that it should be located on the 5-fold axes near the RNA entry and exit channels. To determine the initial location of the RdRP inside the procapsid of bacteriophage 6, we performed cryo-electron microscopy of wild type and mutant procapsids and complemented these data with biochemical determinations of copy numbers. We observe ring-like densities on the 3-fold axes that are strong in a mutant that has 10 copies of P2 per particle; faint in wild type, reflecting the lower copy number of 3; and completely absent in a P2-null mutant. The dimensions and shapes of these densities match those of the known crystal structure of the P2 monomer. We propose that, during maturation, the P2 molecules rotate to occupy positions closer to adjacent 5-fold vertices where they conduct replication and transcription.

Received for publication, December 26, 2007 , and in revised form, February 15, 2008.

^* This work was supported by the Intramural Research Program of NIAMS, National Institutes of Health and by Grant GM34352 (to L. M.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Dept. of Biochemistry and Molecular Genetics, University of Virginia, Box 800733, Charlottesville, VA 22908.

² To whom correspondence should be addressed: Bldg. 50, Rm. 1515, 50 South Dr., MSC 8025, NIH, Bethesda, MD 20892-8025. Tel.: 301-451-8241; Fax: 301-480-7629; E-mail: Bernard_Heymann{at}nih.gov.

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