Studies of promoter recognition and start site selection by T7 RNA polymerase using a comprehensive collection of promoter variants

We have examined the behavior of T7 RNA polymerase (RNAP) at a set of promo ter variants having all possible single base pair (bp) substitutions. The p olymerase exhibits an absolute requirement for initiation with a purine and a strong preference for initiation with GTP vs ATP. Promoter variants that would require initiation at the normal start site (+1) with CTP or UTP res ult in a shift in initiation to +2 (with GTP). However, the choice of start site is little affected by base substitutions elsewhere in the initiation region. Furthermore, when the initiation region is shifted either one nucle otide (nt) closer or 1 nt further away from the binding region, transcripti on still begins the same distance downstream. These results indicate that t he sequence around the start site is of little importance in start site sel ection and that initiation is directed a minimum distance of 5 nt downstrea m from the binding region. At promoters that initiate with +1 GGG, T7 RNAP synthesizes a ladder of poly(G) products as a result of slippage of the tra nscript on the three C residues in the template strand from +1 to +3. At pr omoter variants in which there is an opportunity to form a longer RNA-DNA h ybrid, this G-ladder is enhanced and extended. This observation is not in a greement with recent suggestions that the RNA-DNA hybrid in the initiation complex cannot extend further than 3 bps upstream from the active site [Che etham, G., Jeruzalmi, D., and Steitz, T. A. (1999) Nature 399, 80-83].