Mutational analysis of a satellite phage activator.

Abstract

The late gene activator, Delta, of satellite phage P4 is more efficient than the Delta of satellite phage phiR73 in utilizing a P2 helper prophage that lacks an activator (ogr) gene. Analysis of P4 Delta is complicated by the fact that this protein contains two tandem phiR73 Delta-like domains. We performed a mutational analysis of phiR73 Delta, in order to select mutations that might not be found using P4 Delta. The host RNA polymerase alpha subunit mutation rpoA155 (L289F) blocks the growth of P2, P4, and P4 carrying the delta gene of phiR73. A mutant of this latter phage that can grow in the presence of rpoA155 carries a V19M mutation in phiR73 Delta. This suggests that aa 19 contacts RNA polymerase, in addition to the aa residues 13, 42 and 44, that have been implicated in interactions with RNA polymerase by previous mutational analyses of P2 ogr and P4 delta. In corroboration of the proposed role of the regions at aa residues 19, 42, and 44, we found phiR73 Delta mutations in these regions that showed a reduced activation of late gene expression, but a normal ability to bind to late gene promoters. All activators in the Delta class contain four Cys residues that bind Zn2+. Mutation of these aa residues in phiR73 Delta eliminated late gene activation. Spectroscopic analysis of these mutant proteins revealed that they were unable to bind Zn2+. Histidine residues were substituted for two of the Cys residues (C30 and C35), changing a C2C2 type Zn-binding motif to a C2H2 motif. Although His residues are used in coordinating Zn2+ in other proteins, these His substitutions resulted in complete loss of activity and the inability to bind Zn2+.