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Title: ANALYSIS OF T4 PHAGE CONTAINING FUSION-LENGTHENED FORMS OF THE PORTAL PROTEIN

Author
item Baumann, Richard
item BLACK, LINDSAY - UNIVERSITY OF MARYLAND

Submitted to: Evergreen International Phage Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 7/31/2003
Publication Date: 7/31/2003
Citation: Baumann, R.G., Black, L.W. 2003. Analysis of t4 phage containing fusion-lengthened forms of the portal protein [abstract]. Evergreen International Phage Meeting.

Interpretive Summary:

Technical Abstract: In bacteriophage packaging, DNA is fitted through a dodecameric portal ring (connector) situated at one vertex of the icosahedral prohead. To examine the function of the portal during packaging, we have previously reported assembly of phage T4 that contain fusion-lengthened forms of the portal protein gp20 (1). Three different, gene20 amber(am) mutant phages, as well as 20(am)Hoc(am) double mutants, could produce progeny phage when infecting bacterial cells expressing NH2-or COOH- terminal lengthened portal fusions. Full-length fusions can not solely serve as portal, however fusion proteins can be incorporated into portal dodecamer in varying amounts when co-expressed and co-assembled into dodecamer with wild type, near wild type sized gp20 amber fragments, or near wild-type sized fusion fragments. Carboxy-terminal gp20-GFP (green fluorescent protein) and gp20-HOC (highly antigenic outer capsid protein) fusions in phage were inaccessible to trypsin digestion and likely reside in the interior of the phage, consistent with the f29 portal structure (2). Using 20(am)Hoc(am) phage to infect, trypsin digestion analysis of progeny phage containing NH2-terminal HOC-gp20 fusions, which contain trypsin susceptible arg residues in the linker region, yielded protected forms of both the gp20 and HOC proteins, consistent with protection in wild type phage particles of both gp20 and HOC proteins from trypsin digestion. Analysis of HOC-gp20 fusion proheads also showed protection of the HOC protein from trypsin digestion only after expansion (when the HOC site becomes available). These results are also consistent with portal structure (2), and suggest that the portal protein can be tethered via HOC binding to its capsid binding site before packaging. To improve this analysis, 20(am)Hoc(deletion) (D) phage were constructed (to eliminate HOC protein from infecting phage) and used to infect bacteria expressing the HOC-gp20 fusion. As with 20(am)Hoc(am) infections, trypsinized progeny phage containing HOC-gp20 showed both protected gp20 and HOC proteins. In fact, quantification shows that the levels of protected HOC and gp20 are comparable, suggesting that the fusion proteins are incorporated at the portal position and that the HOC protein is tethered from this position. HOC-gp20 fusion containing proheads were also purified from 16(am)17(am)20(am)Hoc(D) infected bacteria and trypsinization showed protected HOC in expanded proheads. Furthermore, immunogold labeling analysis of these HOC-gp20 proheads using HOC antibody showed localization of the HOC at the apical vertex of the prohead as expected from a portal location. Our data therefore suggest that a packaging competent prohead portal can be tethered either from within (via GFP packed into a head containing 500mg/ml DNA) or without the head (via HOC attached to its capsid binding site). Overall, these results are in conflict with the portal rotation model for DNA translocation into the prohead (2,3). We have recently shown a multimeric form of the T4 terminase large subunit, gp17, has an elevated ATPase activity consistent with the energetic requirement of DNA packaging (4), and favor models for DNA packaging by a packasome complex (portal plus terminase) where ATPase motor proteins drive DNA rotation-coupled translocation into the prohead.