The MTE, a new core promoter element for transcription by RNA polymerase II

This thesis examines the functions of the motif ten element (MTE) as a core promoter motif for transcription by RNA polymerase II. Motif 10 was initially identified as an over-represented sequence that is located downstream of the transcription start site in a biocomputational analysis of Drosophila core promoters. In this study, I determined that the motif 10 sequence contains the MTE, which functions cooperatively with the initiator (Inr) element to promote basal transcription by RNA polymerase II. In addition, the consensus sequence of the MTE was refined as C-G-A/T-G-C-C-G-A/T-G/A-C. Functional characterization of the MTE activity by in vitro transcription experiments revealed that the spacing between the MTE and the Inr is essential for transcriptional activity, and the MTE is precisely located at nucleotide positions +18 to +27 relative to the transcription start site. The MTE functions independently of other core promoter elements such as the downstream promoter element (DPE) and TATA-box motifs, and is able to compensate for the loss of these elements. In addition, the MTE exhibits strong functional synergism with both the TATA-box as well as the DPE. Notably, the functions of the MTE appear to be conserved from Drosophila to humans. DNase I footprinting experiments demonstrated that the MTE is responsible for mediating TFIID binding to the TATA-less, MTE-containing promoters. Further studies revealed that the MTE can act in concert with the DPE in heterologous promoters to greatly enhance core promoter affinity for TFIID. Taken together with the in vitro transcription studies, these results indicate that the transcriptional strength of the core promoters is directly correlated to the strength of the promoter-TFIID interactions, which are mediated, in part, by the MTE. In addition, the results of photo-crosslinking analysis of purified TFIID binding to MTE-containing promoters suggest that TAF6 and TAF9 are localized in proximity to the MTE. Altogether, these studies provide evidence that the MTE functions in the recruitment of TFIID to the TATA-less, MTE-dependent core promoters, to facilitate transcription by RNA polymerase II.