Physicochemical studies on interactions between DNA and RNA polymerase. Isolation and mapping of a T7 DNA fragment containing the early promoters for Escherichia coli RNA polymerase

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Abstract
The cleavage sites in the early promoter region of coliphage T7 were mapped for 4 restriction enzymes. They are, from the left end in base pairs, 1100 and 740 for Hinf; 680, 620, 530, 240, 77 and 67 for Hind II; 620 and 530 for Hpa II; 790 for Alu I. The nucleotide sequence between the Hind II site at 680 base pairs from the left end and the Hinf site at 740 base pairs from the left end was determined, from which the start point of the promoter A3 is located at 720 base pairs from the left end. The start points of the other 2 major promoters A1 and A2 are deduced to be at 460 and 580 base pairs from the left end, respectively, from the chain lengths of the in vitro transcripts of the 1100 base-pairs long Hinf fragment. Similar to the sequences of the pL and pR promoters of phage .lambda. and a sequence in SV-40 used by E. coli RNA polymerase as a promoter, the sequence of the A3 promoter of T7 has a Hind II restriction site .apprx. 30 base pairs upstream to the start point of RNA synthesis. No such Hind II sites exist for the A1 and A2 promoters. Experiments on the protection of some of the restriction sites on the 1100 base-pairs-long Hinf fragment by RNA polymerase binding support the EM observations of others that, besides the 3 sites A1, A2 and A3, there is at least a 4th site at which E. coli RNA polymerase can bind strongly. Besides the Hind II site at 680 base pairs from the left end and the Hinf site at 740 base pairs from the left end, which are presumably protected by the binding of a single RNA polymerase at the A3 site, the Hind II site at 240 base pairs from the left end is protected at a level of 5 polymerase molecules/DNA fragment. The possible existence of several minor promoter sites in the early promoter region, the 3 major promoter sites, is discussed.