Figure 4

Results of experiments showing that RB69 gp32 is an autogenous translational repressor. For Panel A, λCI857PLN-bearing plasmid clones of the diagrammed DNA segments were heat-induced (42°C) and assayed for gp32 synthesis as described in other work [24,27]. RBG32 is a DNA segment that carries the wild-type sequence from -120 through +900 relative to the first base of the initiator AUG of RB69 gene 32. RBG32Δop is a truncated derivative of RBG32 that lacks elements of the putative RNA pseudoknot of RB69 gene 32 (Figs 3 & 6). PL8 is identical to RBG32 except that it carries a single-base substitution (marked with an asterisk) in codon 173, leading to a F173S substitution in RB69 gp32. PL2 is similar to RBG32 and PL8, except that it carries several point mutations (map positions marked with asterisks). Panel B shows results of an experiment in which purified RB69 gp32 was shown to inhibit in vitro translation of purified mRNA from the cloned RBG32 fragment, as well as mRNA from in vitro expressed plasmid clone (coupled transcription/translation). Conditions for these assays are described in METHODS.