Mapping of Class II Promoter Sites Utilized In Vitro by T7-Specific RNA Polymerase on Bacteriophage T7 DNA

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Restriction endonuclease Bgl II cleaves T7 DNA at a unique site (28.76% on the standard T7 map), yielding two fragments of molecular weights 18.9 × 106 (A) and 7.6 × 106 (B). Fragment B, representing the leftmost portion of the genome, has been purified by zone sedimentation. Transcription of fragment B by T7-specific RNA polymerase gives only r-strand-specific RNA. Analysis of the products by polyacrylamide gel electrophoresis reveals four major RNA species which have apparent molecular weights of 2.1 × 106, 1.36 × 106, 0.85 × 106 and 0.125 × 106, respectively. Each of these RNAs is reduced in size when transcription is carried out with fragment B, which has been shortened by treatment with Escherichia coli exonuclease III. Therefore, each of the transcripts must be terminated at the right end of fragment B. Analysis of the molecular weights of the four transcripts produced from whole and from exonucleolytically shortened fragment B suggests that these transcripts are read from promoters located at 13.5, 18.9, 22.6, and 27.9%, respectively, on the standard T7 map. Hence, there are at least four promoters governing the transcription of the class II region. Transcripts initiated at these promoters on intact T7 DNA appear to read through the class II and part of the class III genetic region and terminate at the strong terminator for T7-specific RNA polymerase near 61%. Transcription of fragment B which has been cleaved with the restriction endonuclease Hpa I seems to activate a fifth promoter for T7-specific RNA polymerase. This promoter appears to be identical to the promoter previously described by Oakley and Coleman (Proc. Natl. Acad. Sci. U.S.A. 74:4266-4270, 1977) that maps near 15% on the standard T7 map. Little or no RNA is read from T7 Bgl II fragment B, which has a mobility expected for a transcript read from this promoter. However, upon cleavage with Hpa I, this promoter is utilized approximately 10-fold more efficiently than the other class II promoters. The mechanism of this activation is not yet known.

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