Kinetic and thermodynamic basis of promoter strength: multiple steps of transcription initiation by T7 RNA polymerase are modulated by the promoter sequence.

Transcription initiation by T7 RNA polymerase (T7 RNAP) is regulated by the specific promoter DNA sequence that is classically divided into two major domains, the binding domain (-17 to -5) and the initiation domain (-4 to +6). The occurrence of non-consensus bases within these domains is responsible for the diversity of promoter strength, the basis of which was investigated by studying T7 promoters with changes in the promoter specificity region (-13 to -6) of the binding domain and/or the melting region (-4 to -1) of the initiation domain. The transient state kinetics and thermodynamic studies revealed that multiple steps in the pathway of transcription initiation are modulated by the promoter DNA sequence. Three base changes in the promoter specificity region at -11, -12, and -13, found in the natural phi 3.8 promoter, reduced the overall affinity of the T7 RNAP for the promoter DNA by 2-3-fold and decreased the rate of pppGpG synthesis, the first RNA product. Promoter opening is thermodynamically driven in T7 RNAP, and a single base change in the melting region (TATA to TAAA) decreased the extent of open complex generated at equilibrium. This base change in the melting region also increased the K(d) of (+1) GTP and the dissociation rate of pppGpG. Thus, transcription initiation at various T7 promoters is differentially regulated by initiating GTP concentration. The specificity and melting regions of T7 promoter DNA act both independently and synergistically to affect distinct steps of transcription initiation. Although each step in the initiation pathway is affected to a small degree by promoter sequence variations, the cumulative effect dictates the overall promoter strength.