The T7, T3, and SP6 RNA polymerases recognize very similar,
yet distinct, promoter sequences. The high homology
among the promoter sequences suggests that differential
promoter recognition must derive from relatively small
changes in the protein. Steady state kinetic analyses
of transcription from the T3 consensus promoter and
from promoters modified in the region critical to specific
recognition reveal details concerning which functional
groups contribute to this recognition. Modifications
include base pair substitutions, single base substitutions
(mismatches), and simple functional group modifications
at unique sites in the promoter. The results show that
T3 RNA polymerase recognizes the amino group on the
nontemplate cytidine in the major groove at position
-10, while the identity of the base on the template
strand is less critical to binding. In contrast, recognition
at position -11 allows a greater range of modifications
and seems to have a more complex recognition. The results
do not seem to be consistent with a single recognition
contact at this position, however, some groups may
be ruled out as simple recognition contacts. While
major groove modifications weaken binding at positions
-10 and -11, the removal of an exocyclic amino group
from the minor groove at either position does not disrupt
binding, further supporting a model for promoter recognition
in which the enzyme binds to one face of closed duplex
DNA in this region. The effects of these changes in
the DNA structure on the kinetics of initiation are
compared to complementary results from the T7 system.Return to publication list
