MAPPING THE INITIATION SITES OF IN-VITRO TRANSCRIPTS OF BACTERIOPHAGE-S13

Analysis of in vitro run-off transcripts synthesized by Escherichia co li RNA polymerase holoenzyme on linearized bacteriophage S13 DNA templ ates revealed five major transcription initiation sites. The sites, lo cated at positions 45, 982, 1823 (1827), 4876 and 5211, are each withi n the boundaries of promoters or putative promoters previously mapped by footprinting and RNA polymerase binding analyses. They correspond t o initiations at promoters upstream of the A, B, and D genes, and at a medium-affinity and a high-affinity RNA polymerase binding site P-521 1, respectively. Sequence analysis of the 5'-ends of two transcripts c onfirmed their initiation with pppA at nt 982 and nt 5211, the B gene and high-affinity binding site P-5211, respectively. Some of the trans cripts initiated at nt 4876 and nt 5211 terminated at nt 64, providing direct evidence of the functionality of a p-independent termination s ite at nt 64.