Abstract
Efforts to understand the impact of core promoter architecture on the mechanism of transcription initiation by RNA polymerase II have been hampered by lack of well defined, reconstituted transcription systems responsive both to efficiently transcribed consensus and near consensus TATA box-containing promoters and to considerably weaker TATA-less promoters. In this report, we investigate the influence of core promoter structure on the mechanism of assembly of the RNA polymerase II preinitiation complex using a highly purified, holoTFIID-dependent transcription system that permits sensitive measurement of transcription initiation from a wide variety of TATA and TATA-less promoters in the absence of transactivators. A direct comparison of the requirements for formation of stable preinitiation intermediates at these promoters led to the discovery that, whereas holoTFIID binds avidly to the consensus TATA- and strong initiator-containing adenovirus major late (AdML) promoter to form the first stable intermediate on the pathway leading to formation of the complete preinitiation complex, it binds poorly not only to TATA-less promoters but also to promoters with consensus or near consensus TATA elements. With the exception of the AdML promoter, formation of stable preinitiation intermediates at each of the promoters tested was found to be strongly dependent on RNA polymerase II, holoTFIID, and TFIIB and was stimulated by TFIIF. Based on these observations, we suggest that RNA polymerase II assembles with many TATA and TATA-less promoters by a common pathway.
Highlights
Efforts tounderstand the impact of core promoterar- sequence elements direct the action of at least two classes of chitecture on the mechanism of transcription initiation transcription factors: initiation factorsi,ncludingTFIIA,TFIIB, by RNA polymerase I1 have been hampered by lack of TFIIE, TFIIF, TFIIH, and the TATA factor, TFIID
A direct comparison of the requirements for formation of stable preinitiation intermediates at these promoters led to the discovery that, whereas holoTFIID binds avidly to the consensus TATA- and strong initiator-containingadenovirus major late ( A m ) promoter to form the first stable intermediate on the pathway leading to formaunit TBP), which have been shown to be essential for productive binding of RNA polymerase I1to the core regions of a large number of promoters (1, 21, and DNA binding transcriptional activators, such as the glutamine-rich activator Spl, the proline-rich activator CTFMF1, and the acidic activator GCN4, which are not essential for basal transcription but which mediate the action of upstream promoter elements and enhancers upon RNA polymerase I1 and the initiation factors [3]
Because models for assembly of the preinitiation complex Reconstitution of Basal Danscription from the DHFR Prohave been based on studies of only a limited number of pro- moter-To begin to investigate theinfluence of promoter strucmoters, we have carried out a systematic analysis of the re- ture on formation of the RNA polymerase I1 preinitiation comquirements for formation of stable preinitiation intermediates plex, we wished to compare the requirements for formation of at a variety of consensus TATA and TATA-less promoters
Summary
Because models for assembly of the preinitiation complex Reconstitution of Basal Danscription from the DHFR Prohave been based on studies of only a limited number of pro- moter-To begin to investigate theinfluence of promoter strucmoters, we have carried out a systematic analysis of the re- ture on formation of the RNA polymerase I1 preinitiation comquirements for formation of stable preinitiation intermediates plex, we wished to compare the requirements for formation of at a variety of consensus TATA and TATA-less promoters. Moters whose -30 elements deviate substantiallfyrom the con- Substantial evidence from transfection studies and from in sensus TATA sequence At these promoters, formationof stable vitro transcription experiments carried out in partially fracpreinitiation intermediates depends on the presence of TFIIB tionated extracts indicates that the DNA binding transcripand RNA polymerase I1 in addition thooloTFIID. After 3 min,heparinwas added to prevent further since, once a n initial “committed” complex has formed, subse- rounds of initiation 69-61), andreaction mixtures were incuquent stepsof initiation are resistant chtoallenge by additional bated a n additional 20 min to allow synthesis of full-length promoter DNA In these and all subsequent tempclhaatlelenge runoff transcripts. Both of these “test” templatesdirect synthesis of -250-nucle- core promoter with a t,, > 1h (data not shown)
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