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 changes in the initiation region of the T7 promoter on promoter function. At promoters in which the template strand (T strand) is intact, initiation is directed a minimal distance of 5 nt downstream from the binding region, and if there is a C residue at that position initiation commences at that site with GTP. This effect is observed regardless of the surrounding sequence and whether or not the consensus sequence is present in the non-template (NT) strand. Although the sequence of the DNA surrounding the start site is not critical for start site selection it is important for melting of the promoter and stabilization of the initiation complex. At consensus promoters in which the integrity of T strand is interrupted by nicks or gaps between -5 and -2, the enzyme is able to insert the 3′ end of the interrupted T strand into the active site and to localize the start site at +1 correctly. However, on gapped promoters in which the T strand base at ?1 has been removed initiation occurs not at the C at +1 (which would now be at the 3′ terminus of the T strand) but at the next C, consistent with a need for a base just upstream in the T strand to position the start site. Strikingly, the synthesis of poly(G) products (which arise by transcript slippage) is dramatically reduced or eliminated on nicked or gapped promoters. Furthermore, interrupting the T strand results in a defect in displacement of the nascent RNA and a decrease in the release of abortive initiation products 8-11 nt in length. These effects are attributed to a change in the manner in which the nicked T strand is retained in the initiation complex and a lowered barrier to translocation of the template.