The efficiency of promoter clearance distinguishes T7 class II and class III promoters.
Promoter strength has been defined as the relative production of transcripts from a promoter. For T7 transcription it has frequently been observed that T7 class III promoters are qualitatively stronger than T7 class II promoters. In previous work it was observed that the maximum rates of initiation of three class III and three class II promoters show no class distinctions (Ikeda, R. A., Lin, A. C., and Clarke, J. (1992) J. Biol. Chem. 267, 2640-2649). This suggests that the efficiency of the conversion of the polymerase initiation complex to a stable transcription complex contributes to the overall strength of T7 promoters. The class differences in the strengths of T7 class II and class III promoters are confirmed by measuring the relative synthesis of run-off transcripts. These results show that the relative strengths of the class III promoters, phi 6.5, phi 10, and phi 13, are all comparable ranging from 0.61 for phi 6.5 to 1.00 for phi 10, while the relative strengths of the T7 class II promoters, phi 1.1B, phi 1.3, and phi 3.8, vary widely. One T7 class II promoter, phi 1.1B (relative strength = 0.34), approaches the strength of the class III promoters, while the other T7 class II promoters, phi 1.3 (relative strength = 0.045) and phi 3.8 (relative strength = 0.070) are nearly inactive. The efficiency of promoter clearance is then determined by measuring the relative production of small transcription products in comparison with the production of run-off transcripts. These measurements clearly distinguish the T7 class III promoters from the T7 class II promoters. It is found that 68-75% of all initiations at the T7 class III promoters phi 6.5, phi 10, and phi 13 produce a run-off transcript, while only 16-36% of the initiations at the T7 class II promoters phi 1.1B, phi 1.3, and phi 3.8 produce a run-off transcript. Clearly, promoter clearance contributes to the difference in promoter strengths of the T7 class II and III promoters.
Ikeda
R A
RA
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta 30332-0400.
D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D005814:Genes, Viral; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D013604:T-Phages / Q000201:enzymology / Q000235:genetics*; D013698:Templates, Genetic; D014158:Transcription, Genetic
D004279:DNA, Viral; D012321:DNA-Directed RNA Polymerases