The role of the T7 Gp2 inhibitor of host RNA polymerase in phage development

Abstract

Bacteriophage T7 relies on its own RNA polymerase (RNAp) to transcribe its middle and late genes. Early genes, which include the viral RNAp gene, are transcribed by the host RNAp from three closely spaced strong promoters-A1, A2, and A3. One middle T7 gene product, gp2, is a strong inhibitor of the host RNAp. Gp2 is essential and is required late in infection, during phage DNA packaging. Here, we explore the role of gp2 in controlling host RNAp transcription during T7 infection. We demonstrate that in the absence of gp2, early viral transcripts continue to accumulate throughout the infection. Decreasing transcription from early promoter A3 is sufficient to make gp2 dispensable for phage infection. Gp2 also becomes dispensable when an antiterminating element boxA, located downstream of early promoters, is deleted. The results thus suggest that antiterminated transcription by host RNAp from the A3 promoter is interfering with phage development and that the only essential role for gp2 is to prevent this transcription.

Figure 1. Induction of β-galactosidase activity during wild-type and gp2-deficient T7 infections

Levels of β-galactosidase activity (in Miller units) at various times post-infection. lacZ was induced with 0.5 mM IPTG at the time of phage addition. The multiplicity of infection was 10 and less than 5% of the cells survived the infection. A representative result from four independent experiments is shown.

Figure 2. Primer extension analysis of host and viral transcripts after wild-type T7 infection

A. Wild-type E. coli cells were induced at time 0 with 0.5 mM IPTG. Total RNA was purified from cells collected at the indicated times post-induction and primer extension reactions were carried out. In each right-hand panel, rifampicin (100 µg/ml) was added to induced cells 5 minutes post-induction (indicated by a downward red arrow). Individual primer extension products are labeled at the right hand side of the figure. Two different primers were used to reveal the lacZ transcript, resulting in two different primer extension products. B. As in A but cells were infected with wild-type T7 phage (MOI of 10) at the time of the induction. Additional primers designed to reveal transcripts initiated from the early promoter region of the phage were added to the primer extension reactions. Promoters to which specific primer extension products correspond are identified on the left hand side of the figure. The “ompA*” label identifies an ompA primer-specific extension product that is only seen in infected cells (see text for details). Under the conditions of the experiment, cell lysis occurs 35 minutes post-infection. Less than 5% of the cells survive the infection.

Figure 3. Processing of the ompA transcript during T7 infection

A predicted structure of ompA 5’-UTR between the transcription start point and the initiating codon of the ompA ORF, the T7-induced processing site generating ompA*, and known RNAse E sites indicated. The schematic is based on Fig. 1B of reference .

Figure 4. Primer extension analysis of host and viral transcripts during non-productive infections when the gp2-host RNAP interaction is disrupted

The experiment was conducted as described in the Figure 2 legend. A. Wild-type E. coli cells were infected with T7^2am. B. JE1134 E. coli cells (containing rpoC^{Δ1149–1190}deletion) were infected with the wild-type phage.

Figure 5. Down mutations in the A3 promoter allow productive infection of JE1134 cells

A. T7 A3 promoter mutations that allow productive infections of JE1134 host. The wild type sequence is shown at the top. Promoter consensus elements are labeled and highlighted by color. The transcription start site is shown by a rightward arrow. Sequences of T7 A3 promoters from phages that productively infect JE1134 are shown below. The sites of mutations are highlighted with red color. B. Primer extension analysis of host and viral transcripts during productive infections of wild-type (left panel) and JE1134 (right panel) cells with T7 A3(−35) phage. The experimental design was as described in the Figure 2 legend.

Figure 6. Primer extension analysis of host and viral transcripts during productive infection of JE1134 cells with the T7^boxA phage

The experiment was conducted as described in the Figure 2 legend.