Mapping the initiation sites of in vitro transcripts of bacteriophage S13

Analysis of in vitro run-off transcripts synthesized by Escherichia coli RNA polymerase holoenzyme on linearized bacteriophage S13 DNA templates revealed five major transcription initiation sites. The sites, located at positions 45, 982, 1823 (1827), 4876 and 5211, are each within the boundaries of promoters or putative promoters previously mapped by footprinting and RNA polymerase binding analyses. They correspond to initiations at promoters upstream of the A, B, and D genes, and at a medium-affinity and a high-affinity RNA polymerase binding site P5211, respectively. Sequence analysis of the 5'-ends of two transcripts confirmed their initiation with pppA at nt 982 and nt 5211, the B gene and high-affinity binding site P5211, respectively. Some of the transcripts initiated at nt 4876 and nt 5211 terminated at nt 64, providing direct evidence of the functionality of a p-independent termination site at nt 64.