Inhibition of the Escherichia coli RNA Polymerase by Bacteriophage T7 Gene 2 Protein

Abstract

Allele specificity of the Escherichia coli dnaA gene function in the replication of plasmids derived from bacteriophage λ has been demonstrated previously. Here, using a series of dnaA temperature-sensitive mutants, we investigated dnaA allele specificity of the replication of phages λP+ and λPts1πA66. We found that phage λP+ produces its progeny efficiently at 43°C irrespective of the dnaA allele, whereas λPts1πA66, which is unable to develop lytically in the dnaA+ host at this temperature, can replicate with different efficiency in certain dnaA mutants. Since the main role of DnaA in λ development seems to be stimulation of transcription from the pR promoter, we measured the activity of this promoter (using a pR-lacZ fusion) and the abundance of pR-derived transcripts (by Northern blotting analysis) in dnaA+ host and dnaA(ts) mutants at 30 and 43°C. We found significant differences in the activity of pR in various dnaA(ts) mutants at 30°C, which indicate different levels of stimulation of this promoter by products of particular dnaA alleles at permissive temperature. Differential levels of DnaA-mediated stimulation of pR in various dnaA(ts) mutants were also found at 43°C. Stimulation of the pR promoter by DnaA is necessary for both efficient production of the λ replication proteins, O and P, and effective transcriptional activation of oriλ. The differences in the efficiency of pR activation observed in dnaA mutants at 30 and 43°C can explain the mechanisms of allele specificity of dnaA gene function in the replication of bacteriophage λ and plasmids derived from this phage.