Assembly of Bacteriophage T4 DNA Packaging Motor: Analysis of Portal-Terminase Interactions

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Assembly of Bacteriophage T4 DNA Packaging Motor: Analysis of Portal-Terminase Interactions

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dc.contributor.advisor Rao, Venigalla B en_US Hegde, Shylaja M. en_US
dc.contributor.other Gollin, Johan E en_US
dc.contributor.other Mullins, Michael en_US
dc.contributor.other Barkat, Aaron en_US
dc.contributor.other Dutta, Biprodas en_US 2011-02-24T20:46:58Z 2011-02-24T20:46:58Z 2009 en_US 2011-02-24T20:46:58Z
dc.identifier.other Hegde_cua_0043A_10057 en_US
dc.description Degree awarded: Ph.D. Biology. The Catholic University of America en_US
dc.description.abstract The icosahedral double-stranded DNA bacteriophages and herpes viruses package their genomes into preformed proheads by a powerful ATP driven motor. The packaging motor, an oligomer of gp17 (large terminase) in phage T4, is assembled at the special portal vertex of the empty prohead. The T4 motor is the fastest and most powerful motor reported to date. gp17 has an N-terminal ATPase that powers DNA translocation and a C-terminal translocase that causes DNA movement. The dynamic interactions between the motor (gp17) and the portal (gp20) are however poorly understood. Here, using biochemistry, bioinformatics, structure, and molecular genetics, the site in gp17 that interacts with the dodecameric portal protein is determined. Biochemical and structural studies suggest that the N-terminal domain of gp17 interacts with gp20, and that the stoichiometry of prohead-gp17 complex is five subunits of gp17 to twelve subunits of gp20. Sequence alignments predict that there are two potential portal binding sites in gp17. Mutational studies show that the portal binding site I in the N-terminal domain is critical for function whereas the site II in the C-terminal domain is not critical. Second site suppressors of site I D331Q mutant (temperature sensitive) show a single intragenic mutation in the helix-loop-helix (HLH) of N-terminal sub-domain II, suggesting the importance of this motif in portal interaction. Fitting the X-ray structure of gp17 into the cryo-EM density of portal-motor complex showed the same HLH (amino acids 333-352) in contact with gp20. A peptide corresponding to the HLH motif specifically binds to proheads as well as inhibits DNA packaging in vitro. Swapping of non-conserved residues of the helix, but not the conserved residues of the loop, from T4-family phages relieves the DNA packaging inhibition. Together these data for the first time identify a HLH motif in gp17 that interacts with gp20, leading to models for symmetry mismatch between the packaging motor and the portal as well as implications to the mechanism of viral DNA translocation. en_US
dc.format.extent 110 p. en_US
dc.format.mimetype application/pdf en_US
dc.language eng en_US
dc.publisher The Catholic University of America en_US
dc.subject Biology, Molecular en_US
dc.title Assembly of Bacteriophage T4 DNA Packaging Motor: Analysis of Portal-Terminase Interactions en_US
dc.type Text en_US
dc.type Dissertation en_US

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