The ability of T7 DNA to direct rapid-turnover, single-step addition reactions catalyzed by Escherichia coli RNA polymerase has been investigated. In these reactions a nucleoside triphosphate is added to a dinucleoside monophosphate to form a trinucleoside bisphosphate. Only 6 of the 64 possible combinations of substrates gave rapid-turnover reactions according to our criterion for defining these reactions. The six reactions are C-A + UTP→C-A-U, G-A + UTP→G-A-U, C-A + CTP →C-A-C, U-G + UTP→U-G-U, G-U + UTP→G-U-U, and U-A + CTP→U-A-C. Using restriction fragments, we have assigned four of these reactions to known E. coli RNA polymerase promoters on T7 DNA. C-A-U is synthesized at the A1 promoter, U-A-C at the C promoter, and both U-G-U and G-U-U at the D promoter. The remaining two reactions were shown to occur outside the early region, possibly at the E or other minor promoters. C-A + UTP→ C-A-U is also directed by the A3 promoter, but the reaction is a slow-turnover addition with a rate only one-tenth that of A1-directed C-A-U synthesis. Some of the other 58 combinations of substrates also gave very slow-turnover reactions. One of these slow additions, C-G + CTP →C-G-C, has been assigned to the A2 promoter. Our results indicate that on natural DNA rapid-turnover, single-step addition reactions occur at specific sites, most and possibly all of which are promoters. Not all promoters, however, direct rapid single-step additions. The apparent specificity of rapid-turnover, single-step addition reactions may be explained by the relative binding affinities of RNA polymerase to promoter vs. nonpromoter sites along with the low effective concentration of a given trinucleotide coding sequence on natural DNA. Our observation that the same product (C-A-U) is synthesized at significantly different rates on A1 and A3 promoters indicates that rate variations among single-step addition reactions cannot be solely attributed to differences in product formation or dissociation rates. One or more features of the DNA promoter structure outside of the three nucleotides coding for the ribotrinucleotide product must also affect the efficiency of the reaction. Single-step addition reactions should therefore be useful probes in dissecting the influence of DNA sequence on RNA polymerase binding and promoter behavior.