Three Key Subregions Contribute to the Function of the Downstream RNA Polymerase II Core Promoter

Abstract

The RNA polymerase II core promoter is a diverse and complex regulatory element. To gain a better understanding of the core promoter, we examined the motif 10 element (MTE), which is located downstream of the transcription start site and acts in conjunction with the initiator (Inr). We found that the MTE promotes the binding of purified TFIID to the core promoter and that the TAF6 and TAF9 subunits of TFIID appear to be in close proximity to the MTE. To identify the specific nucleotides that contribute to MTE activity, we performed a detailed mutational analysis and determined a functional MTE consensus sequence. These studies identified favored as well as disfavored nucleotides and demonstrated the previously unrecognized importance of nucleotides in the subregion of nucleotides 27 to 29 (+27 to + 29 relative to A₊₁ in the Inr consensus) for MTE function. Further analysis led to the identification of three downstream subregions (nucleotides 18 to 22, 27 to 29, and 30 to 33) that contribute to core promoter activity. The three binary combinations of these subregions lead to the MTE (nucleotides 18 to 22 and 27 to 29), a downstream core promoter element (nucleotides 27 to 29 and 30 to 33), and a novel “bridge” core promoter motif (nucleotides 18 to 22 and 30 to 33). These studies have thus revealed a tripartite organization of key subregions in the downstream core promoter.

We are very grateful to Uwe Ohler, Tammy Juven-Gershon, Timur Yusufzai, and Sharon Torigoe for critical reading of the manuscript. We thank Chris Benner and Chris Glass (University of California, San Diego, La Jolla, CA) for sharing the Drosophila transcription start site data. We also thank George Kassavetis and E. Peter Geiduschek (University of California, San Diego, La Jolla, CA) for the gift of the AB-dUTP photo-cross-linking reagent.

This work was supported by a grant from the National Institutes of Health (GM041249) to J.T.K.