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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 ChlPchip analyses. We find NC[alpha] 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 TFliB recognition element in core promoter regions. Our data establish the genome-wide basis for general and gene-specific functions of NC2 in mammalian cells. genome-wide | occupancy

Research has been conducted on the negative cofactor 2, the TATA box binding protein and DNA ternary complex. The X-ray structure of this complex has been investigated and the regulatory implications of the negative cofactor 2 heterodimer structure are discussed.

The human general co-factors were discovered during biochemical fractionation of mammalian nuclear extracts in functional in vitro assays. They appear to act in concert with other coactivators that bind tightly to the TATA-binding protein and RNA polymerase II. Several co-factors have been shown to interact with general transcription factors, leading either to activation or repression of transcription. At least one subgroup of co-factors that enhance the effects of activators on transcription are DNA-binding proteins located in the chromatin. In fact, one co-factor, the repressor NC2, is structurally related to histones. The understanding of the molecular interplay of such components of the initiation complex in the chromatin - including general co-factors, other co-factors, general factors and activators - will be a major challenge in the future.

P15, a novel human cofactor protein, is distinct from other TATA-binding protein-associated factors involved in activated RNA polymerase II transcription. Sequencing and cloning analyses of p15 and other functionally related proteins reveal that its amino acid sequences show homology to viral and cellular sequences. Protein kinases regulate the cofactor activity of p15 in HeLa cells, and give rise to different phosphorylated forms of p15. There are functional and constitutional homologies between p15 and viral immediate-early regulatory domains.

The results of research conducted on adenovirus major late core promoter show the presence of an initiation pathway, including the linking of the transcription factor TFIID Cor, the derived subunit, the TATA-linking protein TBP (TFIID tau) to the TATA element, eased by transcription factor TFIIA followed by orderly interactions of other general factors. The orderly addition of the general factors results in TFII-I dependent preinitiation networks differing from those formed with TFIIA. Additionally, TBP linked to an In containing TATA-less promoters recommends the methods for stimulating TATA-less promoters. These analyses provide evidence that functionally different paths can be regulated by specific stimulators or repressors.

In eukaryotic cells, the transcription of genes into mRNAs is characterized by an unrivaled wealth of protein coactivator complexes that regulate the enzyme responsible for transcription, RNA polymerase II. A [...]