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 two-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 show a single
apparent activation energy of ~=26 kcal/mole 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.Return to publication list
