Mutational analysis of a satellite phage activator

The late gene activator, Delta, of satellite phage P4 is more efficient tha n the Delta of satellite phage phi R73 in utilizing a P2 helper prophage th at lacks an activator (ogr) gene. Analysis of P4 Delta is complicated by th e fact that this protein contains two tandem phi R73 Delta-like domains. We performed a mutational analysis of phi R73 Delta, in order to select mutat ions that might not be found using P4 Delta. The host RNA polymerase cc sub unit mutation rpoA 155 (L289F) blocks the growth of PZ, P4, and P4 carrying the delta gene of phi R73. A mutant of this latter phage that can grow in the presence of rpoA 155 carries a V19M mutation in phi R73 Delta. This sug gests 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 p hi R73 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 phi R73 Delta eliminated late gene activ ation. Spectroscopic analysis of these mutant proteins revealed that they w ere 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 C2H 2 motif. Although His residues are used in coordinating Zn2+ in other prote ins, these His substitutions resulted in complete loss of activity and the inability to bind Zn2+ (C) 1998 Elsevier Science B.V. All rights reserved.