Isolation of bacteriophage T4 baseplate proteins P7 and P8 and in vitro formation of the P10/P7/P8 assembly intermediate.
Two bacteriophage T4 proteins, P7 and P8, which are components of the phage baseplate have been purified to apparent homogeneity. P7 and P8 are the protein products of T4 genes 7 and 8. A plasmid has been constructed which contains approximately 5 kilobases of T4 DNA, including genes 7 and 8, under the control of the tac promoter. Induction of Escherichia coli W3110iQ cells containing this plasmid resulted in the production of functional P7 and P8. Standard protein isolation procedures were used to purify both P7 and P8 from extracts of induced cells. In T4-infected cells, these two proteins and P10 interact in a strictly ordered sequential manner (P10 + P7----P10/P7,P10/P7 + P8----P10/P7/P8) to form an intermediate in the baseplate assembly pathway. The three purified proteins assembled in vitro to form a limited number of oligomeric species, as determined by nondenaturing gel electrophoresis. P10 and P7 interacted in vitro to form two assemblies with distinct electrophoretic mobilities, both containing P10 and P7. Addition of P8 to this mixture resulted in the disappearance of both P10/P7 species and the appearance of a single new assembly with a different electrophoretic mobility. These interactions occurred without the addition of any catalyst or cofactors. Isolated P11 appeared to add as predicted to the in vitro-formed complexes without affecting the formation of the two P10/P7 or the single P10/P7/P8 intermediates. Interactions between P7 and P8 in the absence of P10 or interactions between P10 and P8 in the absence of P7 could not be detected. These data indicate that purified P10, P7, and P8 interact in vitro in a manner completely in accord with the published assembly pathway and thus establish a system for further study of the regulation of the formation of this assembly intermediate in vitro.
Plishker
M F
MF
Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston.
Rangwala
S H
SH
Berget
P B
PB
D004591:Electrophoresis, Polyacrylamide Gel; D005814:Genes, Viral; D007118:Immunoassay; D010957:Plasmids; D013604:T-Phages / Q000032:analysis* / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D014764:Viral Proteins