Interrupting the template strand of the T7 promoter facilitates translocation of the DNA during initiation, reducing transcript slippage and the release of abortive products

We have explored the effects of a variety of structural and sequence change s in the initiation region of the phage T7 promoter on promoter function. A t promoters in which the template strand (T strand) is intact, initiation i s directed a minimal distance of 5 nt downstream from the binding region. A lthough the sequence of the DNA surrounding the start site is not critical for correct initiation, it is important for melting of the promoter and sta bilization of the initiation complex. At promoters in which the integrity o f T strand is interrupted by nicks or gaps between -5 and -2 the enzyme con tinues to initiate predominately at +1. However, under these conditions the re is a decrease in the release of abortive products of 8-10 nt, a decrease in the synthesis of poly(G) products (which arise by slippage of the nasce nt transcript), and a defect in displacement of the RNA. We propose that un linking the binding and initiation regions of the T strand changes the mann er in which this strand is retained in the abortive complex, reducing or el iminating the need to pack or "scrunch" the strand into the complex during initiation and lowering a thermodynamic barrier to its translocation. (C) 2 001 Academic Press.