Tests of a Model of Specific Contacts in T7 RNA Polymerase-Promoter
Interactions
Charlie Schick and Craig T. Martin
Biochemistry 34, 666-672, 1995.
The T7, T3, and SP6 RNA polymerases represent a highly
homologous family of enzymes that recognize similarly
homologous promoter DNA sequences. Despite these similarities,
the enzymes are highly specific for their respective
promoters. Studies of mutant RNA polymerases have linked
a specific amino acid residue in the protein to recognition
of bases at positions -11 and -10 in the promoter [Raskin,
et al. (1992) J. Mol. Biol., 506-515]. In kinetic analyses
of transcription from synthetic promoters containing
base analog substitutions, we have recently shown that
at positions -11 and -10 of the T3 promoter, T3 RNA
polymerase recognizes functional groups along the nontemplate
strand wall of the major groove [Schick & Martin, (1993) Biochemistry 32, 4275-4780].
We now extend these
studies to the homologous region of the T7 promoter.
The results confirm extrapolations from the T3 system
and show that T7 RNA polymerase recognizes corresponding
functional groups at positions -11 and -10 of the T7
promoter. The results are consistent with a direct
read-out model for recognition of these bases [Raskin,
et al. (1992) J. Mol. Biol., 506-515], in which the
6-carbonyl and the 7-imino group of the nontemplate
guanine at position -11 and the 6-amino group of the
nontemplate adenine at position -10 of the T7 promoter
are directly involved in binding. The results further
support an overall model for promoter recognition in
which the enzyme binds to one face of the duplex DNA
in this upstream region of the promoter.
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Questions? CMartin@Chem.UMass.edu