Restriction fragment analysis of the temporal program of bacteriophage SP01 transcription and its control by phage-modified RNA polymerases

Abstract

Abstract The temporal program of phage SPO1 gene transcription and its regulation by modifications of RNA polymerase have been examined by using endonuclease restriction fragments of the phage genome as specific hybridization probes. Cells of Bacillus subtilis were pulsed-labeled at various times after infection by wild-type SPO1 or by mutants in regulatory gene 28, 33, or 34. Radioactive RNA from the pulse-labeled bacteria was then hybridized to electrophoretically separated restriction fragments of SP01-DNA. From the patterns of hybridization, we identified DNA fragments that contained early, middle, or late phage genes. To examine the control of phage gene transcription by modifications of RNA polymerase, RNA was copied in vitro from uncut SP01-DNA by either the unmodified B. subtilis transcriptase or by two forms of modified RNA polymerase that contained phage-coded regulatory subunits. RNA generated in vitro by each of the RNA polymerases exhibited a distinctive pattern of hybridization to the separated fragments of SPO1-DNA. RNA synthesized by unmodified host enzyme containing sigma factor preferentially annealed to DNA fragments containing early sequences, while RNA copied by the modified forms of RNA polymerase containing the gene 28 product or the products of genes 33 and 34 preferentially annealed to fragments containing middle and late genes, respectively. These findings confirm earlier reports indicating that the temporal program of SPOT gene expression is controlled by phage specified modifications in the transcriptional specificity of B. subtilis RNA polymerase.