IDENTIFICATION, CLONING AND CHARACTERIZATION OF 3 LATE PROMOTERS AT 14.6, 14.8 AND 15.9 PERCENT OF T7 DNA

Several segments of the 12.05 to 18.19% region of bacteriophage T7 DNA were cloned. The recombinant plasmids were assayed for their capacity to support transcription with a cell-free system specific for T7 DNA. A clone (pRW307) of the 12.05% to 16.0% region stimulated RNA synthesis several hundred fold over the vector back ground but a clone (pRW308) of the 16.1 to 18.19% region had only background activity. Clones of two contiguous subfragments of the insertion in pRW307 spanning the regions 12.05 to 14.65% (pRW315) and 14.65 to 16.0% (pRW314) stimulated RNA synthesis to the same level as found for T7 DNA. In contrast, two clones of subfragments of the insertion in pRW315 spanning the regions 12.05 to 12.15% (pRW312) and 12.70 to 14.10% (pRW313) had background activity. Restriction fragments spanning the regions 12.05 to 14.65% and 14.65 to 16.0% were used as templates for highly purified T7 RNA polymerase and the sizes of the discrete RNA products were determined by gel electrophoresis. The results of these experiments indicated that three late, class II promoters exist inside the 12.05 to 18.19% region of T7 DNA; they are located at 14.6, 14.8 and 15.9%. Their sequences, containing a 22 base-pair homologous region, are reported elsewhere (Panayotatos & Wells, 1979b). No evidence for promoters at 13.5 or 14.2% was found, in contrast to conclusions drawn by previous workers. Determination of the DNA sequence of the 14.2% region failed to detect homology with the sequence of the promoters at 14.6, 14.8 and 15.9%. © 1979.