Kinetic analysis of T7 RNA polymerase transcription initiation from promoters containing single-stranded regions

Abstract

T7 RNA polymerase is highly specific for the initiation of transcription from a relatively small consensus promoter sequence. Previous footprinting studies suggested that the enzyme binds specifically to a fully closed duplex form of the promoter, recognizing functional groups along one face of the helix [Muller, D. K., Martin, C. T., & Coleman, J. E. (1989) Biochemistry 28, 3306-3313]. Steady-state kinetic analysis of oligonucleotide-based promoters shows that removal of the nontemplate strand completely within the message region of the DNA (positions +1 through +5) results in no change in binding (as reflected in the parameter Km) and a 2-fold increase in kinetics (as reflected in kcat). Further deletion of the nontemplate strand as far upstream as position -4 has no effect on binding, and although deletion upstream through position -6 weakens binding, specific initiation continues at a high rate. The temperature dependence of the initiation kinetics shows a single apparent activation energy of approximately 26 kcal/mol for the fully duplex promoter. Similar measurements on the promoter lacking the nontemplate strand in the message region show that less than 10% of this barrier is related to melting of the downstream region of the promoter. These results lead us to revise the previous model for recognition to include specific binding to a form of the promoter which is duplex upstream of about position -6 and melted downstream through the start site. Within the melted region, the polymerase interacts significantly only with the template strand of the promoter DNA.