Abstract
The general transcription factor TFIID is a core promoter selectivity factor that recognizes DNA sequence elements and nucleates the assembly of a pre-initiation complex (PIC). The mechanism by which TFIID recognizes the promoter is poorly understood. The TATA-box binding protein (TBP) is a subunit of the multi-protein TFIID complex believed to be key in this process. We reconstituted transcription from highly purified components on a ribosomal protein gene (RPS5) and discovered that TFIIDΔTBP binds and rearranges the promoter DNA topology independent of TBP. TFIIDΔTBP binds ~200 bp of the promoter and changes the DNA topology to a larger extent than the nucleosome core particle. We show that TBP inhibits the DNA binding activities of TFIIDΔTBP and conclude that the complete TFIID complex may represent an auto-inhibited state. Furthermore, we show that the DNA binding activities of TFIIDΔTBP are required for assembly of a PIC poised to select the correct transcription start site (TSS).
Highlights
About sixty proteins, including RNA polymerase II (Pol II) and the general transcription factors (GTFs), assemble at each promoter prior to each round of transcription [1,2]
We showed that TATA-box binding protein (TBP) alone binds RPS5, forming at least two specific complexes with one or more TBP molecules bound, which agrees with previously published DNAse I footprinting of TBP on the RPS5 promoter [8] (Supplementary Figure S3)
There was no complete shift at 20 nM of TBP-associated factors (TAFs) in the presence of 40 nM of TBP, but rather a smear of the free DNA label, which we interpret as free TBP binding to the probe
Summary
About sixty proteins, including RNA polymerase II (Pol II) and the general transcription factors (GTFs), assemble at each promoter prior to each round of transcription [1,2]. Most (~80%) Pol II promoters do not contain a canonical TATA-box [3,4,5,6] These TATA-less genes are constitutively transcribed and frequently involved in “housekeeping” processes, essential for all basic cellular maintenance. The specific DNA binding activity of TBP appears not to be important for the transcription of “housekeeping” genes with TATA-less promoters. We tested the hypothesis that the TAF complex (TFIID∆TBP) recognizes the TATA-less promoter of the ribosomal protein 5 gene (RPS5) independent of TBP in vitro. TFIID may represent an auto-inhibited state, incapable of binding the promoter and rearranging its topology Instead, it is the TBP-lacking TAF complex that binds the promoter and rearranges its topology, thereby providing the nucleation point for assembly of a PIC poised to select the correct TSS. We propose that the TAFs–DNA rather than the TBP–DNA complex represents the nucleation point for PIC assembly on the TATA-less RPS5 promoter
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