Modulation of the Packaging Reaction of Bacteriophage T4 Terminase by DNA Structure

Bacteriophage terminases package DNA through the portal ring of a procapsid during phage maturation. We have probed the mechanism of the phage T4 large terminase subunit gp17 by analyzing linear DNAs that are translocated in vitro. Duplex DNAs of random sequence from 20 to 500 bp were efficiently pa...

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Bibliographic Details
Published in:Journal of molecular biology 2008-08, Vol.381 (1), p.61-72
Main Authors: Oram, Mark, Sabanayagam, Chandran, Black, Lindsay W.
Format: Article
Language:English
Subjects:
Bacteria
bacteriophage
DNA - chemistry
DNA - metabolism
DNA packaging
Endodeoxyribonucleases - metabolism
fluorescence correlation spectroscopy
helicase
Nucleic Acid Conformation
Phage T4
Protein Subunits - metabolism
Spectrometry, Fluorescence
Substrate Specificity
terminase
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Summary:Bacteriophage terminases package DNA through the portal ring of a procapsid during phage maturation. We have probed the mechanism of the phage T4 large terminase subunit gp17 by analyzing linear DNAs that are translocated in vitro. Duplex DNAs of random sequence from 20 to 500 bp were efficiently packaged. Dye and short, single-stranded end extensions were tolerated, whereas 20-base extensions, hairpin ends, 20-bp DNA–RNA hybrid, and 4-kb dsRNA substrates were not packaged. Molecules 60 bp long with 10 mismatched bases were translocated; substrates with 20 mismatched bases, a related D-loop structure, or ones with 20-base single-strand regions were not. A single nick in 100- or 200-bp duplexes, irrespective of location, reduced translocation efficiency, but a singly nicked 500-bp molecule was packaged as effectively as an unnicked control. A fluorescence-correlation-spectroscopy-based assay further showed that a 100-bp nicked substrate did not remain stably bound by the terminase–prohead. Taken together, two unbroken DNA strands seem important for packaging, consistent with a proposed torsional compression translocation mechanism.
ISSN:0022-2836
1089-8638
DOI:10.1016/j.jmb.2008.05.074