NC2 mobilizes TBP on core promoter TATA boxes.

The general transcription factors (GTFs) of eukaryotic RNA polymerase II, in a process facilitated by regulatory and accessory factors, target promoters through synergistic interactions with core elements. The specific binding of the TATA box-binding protein (TBP) to the TATA box has led to the assumption that GTFs recognize promoters directly, producing a preinitiation complex at a defined position. Using biochemical analysis as well as biophysical single-pair Förster resonance energy transfer, we now provide evidence that negative cofactor-2 (NC2) induces dynamic conformational changes in the TBP-DNA complex that allow it to escape and return to TATA-binding mode. This can lead to movement of TBP along the DNA away from TATA.

Global distribution of negative cofactor 2 subunit-alpha on human promoters.

Negative cofactor 2 (NC2) forms a stable complex with TATA-binding protein (TBP) on promoters in vitro. Its association with TBP prevents the binding of TFIIB and leads to inhibition of preinitiation complex formation. Here, we investigate the association of NC2 subunit-alpha with human RNA polymerase II promoter regions by using gene-specific ChIP and genome-wide promoter ChIPchip analyses. We find NC2alpha associated with a large number of human promoters, where it peaks close to the core regions. NC2 occupancy in vivo positively correlates with mRNA levels, which perhaps reflects its capacity to stabilize TBP on promoter regions. In single gene analyses, we confirm core promoter binding and in addition map the NC2 complex to enhancer proximal regions. High-occupancy histone genes display a stable NC2/TFIIB ratio during the cell cycle, which otherwise varies markedly from one gene to another. The latter is at least in part explained by an observed negative correlation of NC2 occupancy with the presence of the TFIIB recognition element in core promoter regions. Our data establish the genome-wide basis for general and gene-specific functions of NC2 in mammalian cells.

Efficient binding of NC2.TATA-binding protein to DNA in the absence of TATA.

Negative cofactor 2 (NC2) forms a stable complex with TATA-binding protein (TBP) on promoters. This prevents the assembly of transcription factor (TF) IIA and TFIIB and leads to repression of RNA polymerase II transcription. Here we have revisited the interactions of NC2.TBP with DNA. We show that NC2.TBP complexes exhibit a significantly reduced preference for TATA box sequences compared with TBP and TBP.TFIIA complexes. In chromatin immunoprecipitations, NC2 is found on a variety of human TATA-containing and TATA-less promoters. Substantial amounts of NC2 are present in a complex with TBP in bulk chromatin. A complex of NC2.TBP displays a K(D) for DNA of approximately 2 x 10(-9) m for a 35-bp major late promoter oligonucleotide. While preferentially recognizing promoter-bound TBP, NC2 also accelerates TBP binding to promoters and stabilizes TBP.DNA complexes. Our data suggest that NC2 controls TBP binding and maintenance on DNA that is largely independent of a canonical TATA sequence.