Kinetic and Thermodynamic Basis of Promoter Stength: Multiple Steps of Transcription Initiation by T7 RNA Polymerase Are Modulated by the Promoter Sequence

摘要

Transcription initiation by T7 RNA polymerase (T7 RNAP) is regulated by the specific promoter DNA sequence that is classically deivided into two major domains, the binding domain (-17 to -5) and the initiation domain (-4 to +6). The occurrence of nonconsensus bases within these domains is repsonsible 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 NRAP for the promoter DNA by 2-3-fold and decreased the rate of pppGpG synthesis, the first RNAa product. Promoter opening is thermodynamically driven in T7 RNAP, and a single base change int he melting region (TATA to TAAA) decreased the extent of open compelx generated at equilibrium. This base change int he melting region also increased the K_d of (+1) GTP and the dissociation rate of pppGpG. Thus, transcription intiaition at various T7 promoters is differentially regulated by initiating GTP concentration. The specificity and melting regions of T7 promoter DNA act both independently and syntergiestically 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.