Kinetic analysis of T7 RNA polymerase transcription initiation from promoters containing single-stranded regions.
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.
Maslak
M
M
Department of Chemistry, University of Massachusetts, Amherst 01003.
Martin
C T
CT
D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013698:Templates, Genetic; D014158:Transcription, Genetic
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D012321:DNA-Directed RNA Polymerases