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

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Abstract
Restriction endonuclease Bgl II cleaves T7 DNA at a unique site (28.76% on the standard T7 map), yielding 2 fragments of MW 18.9 .times. 106 (A) and 7.6 .times. 106 (B). Fragment B, representing the leftmost portion of the genome, was 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 4 major RNA species of apparent MW 2.1 .times. 106, 1.36 .times. 106, 0.85 .times. 106 and 0.125 .times. 106, respectively. Each of these RNA is reduced in size when transcription is carried out with fragment B 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 MW of the 4 transcripts produced from whole and 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 4 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 cleaved with the restriction endonuclease Hpa I seems to activate a 5th promoter for T7-specific RNA polymerase. This promoter appears to be identical to the promoter previously described by Oakley and Coleman 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. 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.