Tests of a Model for Promoter Recognition by T7 RNA
Polymerase: Thymine Methyl Group Contacts
Maribeth
Maslak, Martha D. Jaworski, and Craig T. Martin
Biochemistry
32, 4270-4274, 1993.
The DNA dependent RNA polymerase from bacteriophage
T7 is highly specific for a 17 base promoter sequence.
Interactions between T7 RNA polymerase and its promoter
DNA have been probed using modified oligonucleotides
and a steady state kinetic assay. The incorporation
of deoxyuridine in place of thymidine at individual
sites in the promoter sequence results in the replacement
of an exocyclic methyl group by hydrogen (effectively
removing the thymine methyl). This substitution has
been placed individually at each of the thymines in
the T7 consensus promoter. Many of these substitutions
do not affect binding or catalysis, however, the thymine
methyl group at position -6 is critical to recognition.
The kinetic parameter Km increases approximately ten-fold
while kcat is only slightly affected, suggesting that
this thymine methyl is critical to binding specificity,
but not to the kinetics of initiation. Two methyl
groups near the start site on the template strand (at
positions -1 and -3) also contribute to promoter specificity,
while nearby methyl groups on the nontemplate strand
do not. The implications of these results are discussed
with respect to recent models for promoter binding.
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