The 174-base bacteriophage ?29 prohead RNA (pRNA) is an essential component of the motor that packages the 19-kilobase pair genomic DNA-gp3 complex (DNA-gp3) into the viral precursor capsid (prohead). This motor is one of the strongest molecular motors known, generating ~110 pN of force. pRNA forms a novel cyclic hexamer by intermolecular base pairing of identical molecules. This multimer binds to the head-tail connector of the prohead, where it appears as a pentameric ring by cryoEM-3D reconstruction. A multimer of the ATPase gp16 then binds to the pRNA oligomer to constitute the ATP-hydrolyzing subunits of the motor. pRNA is hypothesized to function in docking of the DNA-gp3 on the prohead, in recognition of the left end of DNA-gp3 to initiate packaging, and in assembly and enzyme function of the DNA translocating ATPase. pRNA exits the DNA-filled head during neck and tail assembly, and it is not a part of the mature virion. Study of the structure and function of this RNA-dependent DNA packaging motor may have general significance in uncovering targets for antiviral agents. The ultimate goal of the research is to determine the structural and functional roles of pRNA in the mechanism of DNA translocation. The current aims are: 1) X-ray crystallography will be used to determine atomic structures of monomeric and oligomeric pRNAs;2) NMR will be used to determine the structure of the pRNA intermolecular pseudoknot and the essential CCA bulge of the A-helix;3) Solution and site-directed hydroxyl radical probing will be used to map pRNA nucleotides in prohead/pRNA complexes with and without the ATPase gp16;4) Site-directed and random pRNA mutants will be isolated to interrogate function of the pRNA in DNA translocation;and 5) Single-molecule laser tweezers studies of pRNA mutants having partial function will probe the roles of pRNA in ?29 DNA translocation.

Public Health Relevance

There is a paucity of antiviral agents, and the design of new drugs depends upon a thorough understanding of the basic mechanisms of viral infection and assembly. The infection and assembly mechanisms of the bacteriophage ?29 serve as models for understanding the infection and assembly mechanisms of dsDNA viruses such as herpesvirus and adenovirus, which have significant commonalities to ?29. The assembly processes of ?29 studied here, such as DNA packaging, are targets for antiviral agents.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Prokaryotic Cell and Molecular Biology Study Section (PCMB)
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Flicker, Paula F
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University of Minnesota Twin Cities
Schools of Dentistry
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Morais, Marc C (2016) Breaking the symmetry of a viral capsid. Proc Natl Acad Sci U S A 113:11390-11392
Mao, Huzhang; Saha, Mitul; Reyes-Aldrete, Emilio et al. (2016) Structural and Molecular Basis for Coordination in a Viral DNA Packaging Motor. Cell Rep 14:2017-2029
Zhao, Wei; Jardine, Paul J; Grimes, Shelley (2015) An RNA Domain Imparts Specificity and Selectivity to a Viral DNA Packaging Motor. J Virol 89:12457-66
Liu, Shixin; Chistol, Gheorghe; Hetherington, Craig L et al. (2014) A viral packaging motor varies its DNA rotation and step size to preserve subunit coordination as the capsid fills. Cell 157:702-713
Cao, Sheng; Saha, Mitul; Zhao, Wei et al. (2014) Insights into the structure and assembly of the bacteriophage 29 double-stranded DNA packaging motor. J Virol 88:3986-96
Nam, Ki Hyun; Haitjema, Charles; Liu, Xueqi et al. (2012) Cas5d protein processes pre-crRNA and assembles into a cascade-like interference complex in subtype I-C/Dvulg CRISPR-Cas system. Structure 20:1574-84
Zhao, Wei; Saha, Mitul; Ke, Ailong et al. (2012) A three-helix junction is the interface between two functional domains of prohead RNA in 29 DNA packaging. J Virol 86:11625-32
Nam, Ki Hyun; Ding, Fran; Haitjema, Charles et al. (2012) Double-stranded endonuclease activity in Bacillus halodurans clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas2 protein. J Biol Chem 287:35943-52
Harjes, Elena; Kitamura, Aya; Zhao, Wei et al. (2012) Structure of the RNA claw of the DNA packaging motor of bacteriophage ýý29. Nucleic Acids Res 40:9953-63
Chistol, Gheorghe; Liu, Shixin; Hetherington, Craig L et al. (2012) High degree of coordination and division of labor among subunits in a homomeric ring ATPase. Cell 151:1017-28

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