Background: Transcription by RNA polymerase II in eukaryotic cells requires the ordered assembly of general transcription factors on the promoter to form a preinitiation complex. Transcriptional activator proteins (activators) stimulate transcription by increasing the rate and/or extent of preinitiation complex assembly. We have shown previously that acidic activators increase the stable association of TFIIB on the promoter, a process we refer to as 'recruitment'. In this study, we provide evidence that diverse activators facilitate TFIIB assembly by a related mechanism. We then investigate the activator-mediated assembly of TFIIB with regard to two aspects of transcription: the distance-dependence of activator function, and reinitiation.
Results: We have previously described amino-acid-substitution mutants of TFIIB that are able to support an activator-independent basal level of transcription but do not respond to acidic activators. We now show that these mutants also do not respond to other classes of activators. We demonstrate that this defect is due to a failure of the activators to recruit the mutant TFIIB to the promoter. Activators often lose activity as their distance from the initiation site is increased. We show that this impaired transcriptional activity correlates with a decrease in TFIIB recruitment. Finally, we find that following the initiation of transcription, TFIIB dissociates from the promoter, requiring the activator-mediated reassembly of TFIIB in the preinitiation complex for each new round of transcription.
Conclusion: We have provided evidence that diverse activators recruit TFIIB to the promoter by a related mechanism. This central step in transcriptional activation is sensitive to promoter architecture, and is required for each new round of transcription.