Weakening of the T7 promoter-polymerase interaction facilitates promoter release

Abstract

During transcription initiation, RNA polymerases retain interactions with their promoters until the RNA is extended to 8-13 nucleotides, at which point the polymerase releases the promoter and moves downstream. It has been shown that release of the T7 promoter is inhibited when the T7 RNA polymerase-promoter interaction is strengthened. Here we asked whether release would be facilitated when the T7 promoter-polymerase interaction is weakened by the introduction of promoter mutations known to reduce promoter activity. Using chemical and enzymatic probes to monitor the position of the polymerase as a function of RNA length, we found that promoter mutations upstream of -4 facilitated release of the polymerase from the promoter, but more downstream mutations did not have such effects. We also found that released complexes turn over more slowly than promoter-bound complexes, indicating that retention of promoter interactions contributes to the dissociation of short RNAs during initial transcription.