Contributions of the TATA box sequence to rate-limiting steps in transcription initiation by RNA polymerase II

We have examined the role of the TATA box in determining transcription initiation frequency in vitro by studying a collection of promoters containing different TATA sequences in the context of the adenovirus major late promoter. In addition to measuring transcription rates, we have determined how the sequence changes affected the association and dissociation kinetics and the affinity of TBP binding. We observed that transcription from promoters containing the highest affinity TATA boxes is limited by the rate with which TBP associates with the promoter. In contrast, transcription from promoters containing lower affinity TATA boxes appears to be limited both by how much TBP is bound and by the relatively low occupancy of the conformation that can undergo subsequent steps in preinitiation complex assembly. The implications of these results in understanding the mechanism of transcription enhancement by transcriptional activators is discussed.