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 promoter variants having all possible single base pair (bp) substitutions. The polymerase 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 result 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 nucleotide (nt) closer or 1 nt further away from the binding region, transcription still begins the same distance downstream. These results indicate that the sequence around the start site is of little importance in start site selection and that initiation is directed a minimum distance of 5 nt downstream 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 transcript on the three C residues in the template strand from +1 to +3. At promoter variants in which there is an opportunity to form a longer RNA-DNA hybrid, this G-ladder is enhanced and extended. This observation is not in agreement with recent suggestions that the RNA-DNA hybrid in the initiation complex cannot extend further than 3 bps upstream from the active site [Cheetham, G., Jeruzalmi, D., and Steitz, T. A. (1999) Nature 399, 80-83].