The 174-base bacteriophage phi29 prohead RNA (pRNA) is essential for in vitro packaging of the 19-kilobase pair DNA-gp 3 complex (DNA-g3) into the viral precursor capsid (prohead). pRNA is an integral part of the phi29 DNA packaging motor, one of the strongest molecular motors characterized, pRNA forms a novel cyclic hexamer by intermolecular base pairing of identical molecules. This multimer binds to the head-tail connector of the prohead, the core of the packaging motor, where it appears as a pentameric ring by cryoEM 3-D reconstruction. A multimer of the packaging ATPase gp16 then binds the pRNA to complete the motor. pRNA is hypothesized to function in docking of the DNA-gp3 and the prohead, in recognition of the left end of DNA-gp3 to initiate packaging, and as a component 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 for assembly of other viruses, including mammalian viruses. The ultimate goal of the research is to determine the structures and the mechanisms by which pRNA constitutes the phi29 DNA packaging motor to catalyze DNA-gp3 translocation into the prohead.
The aims of the current project are to: 1) study the DNA packaging motor comprised of the connector, pRNA and the ATPase gp16 by cryoEM-3D reconstruction and X-ray crystallography; 2) determine the structure of the pRNA intermolecular pseudoknot by NMR; 3) study the packaging motor step size and coordination of pRNA-gp16 motor subunits by the use of laser optical tweezers; 4) determine the role of the N-terminus of gp 10 (connector) in binding pRNA; and 5) identify the DNA packaging RNAs of phages SPP1 and lambda.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM059604-06
Application #
6914999
Study Section
Special Emphasis Panel (ZRG1-MBC-2 (01))
Program Officer
Basavappa, Ravi
Project Start
1999-08-01
Project End
2008-02-29
Budget Start
2005-03-01
Budget End
2006-02-28
Support Year
6
Fiscal Year
2005
Total Cost
$327,474
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Dentistry
Type
Schools of Dentistry
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
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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