Regulation of expression of cloned bacteriophage T4 late gene 23.
The parameters governing the activity of the cloned T4 gene 23, which codes for the major T4 head protein, were analyzed. Suppressor-negative bacteria carrying wild-type T4 gene 23 cloned into plasmid pCR1 or pBR322 were infected with T4 gene 23 amber phage also carrying mutations in the following genes: (i) denA and denB (to prevent breakdown of plasmid DNA after infection) and (ii) denA, denB, and, in addition, 56 (to generate newly replicated DNA containing dCMP) and alc/unf (because mutations in this last gene allow late genes to be expressed in cytosine-containing T4 DNA). Bacteria infected with these phage were labeled with (14)C-amino acids at various times after infection, and the labeled proteins were separated by one-dimensional gel electrophoresis so that the synthesis of plasmid-coded gp23 could be compared with the synthesis of other, chromosome-coded T4 late proteins. We analyzed the effects of additional mutations that inactivate DNA replication proteins (genes 32 and 43), an RNA polymerase-binding protein (gene 55), type II topoisomerase (gene 52), and an exonuclease function involved in recombination (gene 46) on the synthesis of plasmid-coded gp23 in relation to chromosome-coded T4 late proteins. In the denA:denB:56:alc/unf genetic background, the phage chromosome-borne late genes followed the same regulatory rules (with respect to DNA replication and gp55 action) as in the denA:denB genetic background. The plasmid-carried gene 23 was also under gp55 control, but was less sensitive than the chromosomal late genes to perturbations of DNA replication. Synthesis of plasmid-coded gp23 was greatly inhibited when both the type II T4 topoisomerase and the host's DNA gyrase are inactivated. Synthesis of gp23 was also substantially affected by a mutation in gene 46, but less strongly than in the denA:denB genetic background. These observations are interpreted as follows. The plasmid-borne T4 gene 23 is primarily expressed from a late promoter. Expression of gene 23 from this late promoter responds to an activation event which involves some structural alteration of DNA. In these respects, the requirements for expressing the plasmid-borne gene 23 and chromosomal late genes are very similar (although in the denA:denB:56:alc/unf genetic background, there are significant quantitative differences). For the plasmid-borne gene 23, activation involves the T4 gp46, a protein which is required for DNA recombination. However, for the reasons presented in the accompanying paper (Jacobs et al., J. Virol. 39:31-45, 1981), we conclude that the activation of gene 23 does not require a complete breakage-reunion event which transposes that gene to a later promoter on the phage chromosome. Ways in which gp46 may actually be involved in late promoter activation on the plasmid are discussed.
D003001:Cloning, Molecular; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli; D005786:Gene Expression Regulation; D005814:Genes, Viral; D009154:Mutation; D010957:Plasmids; D013604:T-Phages / Q000235:genetics* / Q000378:metabolism; D014764:Viral Proteins / Q000096:biosynthesis
D014764:Viral Proteins; D012321:DNA-Directed RNA Polymerases; D004264:DNA Topoisomerases, Type I