Kinetic Analysis of T7 RNA Polymerase-Promoter Interactions with Small Synthetic Promoters

Craig T. Martin and Joseph E. Coleman, Biochemistry 26, 2690-2696, 1987

Specific interactions between T7 RNA polymerase and its promoter have been studied by a
simple steady-state kinetic assay using synthetic oligonucleotide promoters that produce a short five-base
message. A series of promoters with upstream lengths extending to promoter positions -19, -17, -14, and
-12 show that promoters extending to -19 and -17 produce very specific transcripts with initiation rate
constant kcat = 50 min-1 and a Michaelis constant Kd = 0.02 µM, indicating that the consensus sequence
to position -17 is sufficient for maximum promoter usage. Shortening the upstream region of the promoter
to -14 substantially increases Kd (0.3 µM) but does not significantly reduce the maximum velocity (kcat
= 30 min-‘). Finally, truncation of the promoter at position -12 results in extremely low levels of specific
transcription. The coding and noncoding strands appear to make different contributions to promoter
recognition. Although the double-stranded promoter of upstream length -12 is very poor as a transcription
template, extension of only the noncoding strand to -17 very significantly improves both kcatand Km. In
contrast, extension of only the coding strand results in no significant improvement. Substitution of an AT
base pair at position -10 by CG (as found in T3 RNA polymerase promoters) produces a l0-fold increase
in Km, with little effect on kcat. Comparison of two promoters containing a base pair mismatch at this site
(AG or CT) demonstrates that promoter recognition is very sensitive to the nature of the base on the noncoding
strand and is only slightly affected by the presence of a mismatch created by a wrong base in the coding
strand. These results lead to a model for transcription initiation by T7 RNA polymerase in which promoter
recognition occurs in the melted form of the DNA and in which upstream determinants on the noncoding
strand provide significant specific interactions with the protein.