Localization of the leftmost initiation site for T7 late transcription, in vivo and in vitro

T7 RNA polymerase transcribes the late region of the T7 genome in vitro and in vivo. The strongest leftmost initiation site on the T7 DNA for late transcription has been localized by a multistep hybridization enrichment technique utilizing both in vivo and in vitro late T7 mRNA and T7 DNA with deletions from 2.9 to 7.0% and from 15.2 to 23.5% from the left end of the T7 DNA. These hybridization results are compared to electron microscopic heteroduplex analysis of hybrids constructed from T7 right-strand DNA and total late T7 in vitro RNA. Both methods yield the conclusion that the strongest leftmost initiation site for T7 RNA polymerase lies to the left of gene 1.3, at about 15% from the left end of the T7 DNA. In addition, from the heteroduplex analysis of the in vitro T7 late RNA and the T7 right-strand DNA, it is apparent that the total late region is transcribed in vitro by T7 RNA polymerase.