Recognition of the core promoter by the transcription machinery is essential for correct positioning and assembly of Pol II and the general factors. Sequence elements found in core promoters include the TATA element (TBP binding site), BRE (TFIIB recognition element), Inr (initiator element), and DPE (downstream promoter element)20
. Most promoters contain one or more of these elements, but there is no one element that is absolutely essential for promoter function. The promoter elements are binding sites for subunits of the transcription machinery and serve to orient the transcription machinery at the promoter asymmetrically to direct unidirectional transcription.
The core domain of TBP consists of two imperfect repeats forming a saddle shaped molecule that binds the widened minor groove of an 8-bp TATA element, unwinding about a third of a helical turn and bending the DNA about 80 degrees toward the major groove21,22
). At TATA containing promoters, formation of this protein-DNA complex is the initial step in assembly of the transcription machinery. While the TBP molecule is symmetrically shaped, the protein surface of the two repeats is very divergent, forming a large asymmetric protein-DNA interface, creating a platform for binding other components of the transcription machinery. Biochemical studies elegantly showed that TBP does not bind to TATA elements with high orientation specificity23
, leading to the finding that other promoter elements in combination with TATA determine the orientation of transcription machinery assembly at a promoter. The BRE element was first recognized as a sequence contributing to high affinity binding of TFIIB and TFB to the human and archaeal TBP-DNA complex24,25
. In archaea, where the DNA binding surface of the two TBP imperfect repeats is more symmetrical compared to eukaryotic TBPs, the BRE is the primary determinant of transcription orientation26,27
Figure 2 General transcription factor structures. (a) Structure of TBP (green) bound to TATA-DNA with B-form DNA (grey and red) modeled upstream and downstream of the TATA box21,22. (b) Structure model of the TBP-TFIIA-TFIIB-DNA complex. TBP (green) is shown from (more ...)
The other two core promoter elements with proven function, Inr and DPE likely serve as binding sites for the TAF (TBP Associated Factor) subunits of the general factor TFIID. A combination of two TAFs (TAF1, 2) was found to specifically bind the Inr, and selection for an optimal TAF1-2 binding sequence led to identification of a sequence closely resembling the Inr element28
. Additionally, UV crosslinking has shown that TAF1 and TAF2 are normally positioned close to the Inr29
and that TAF6 and TAF9 lie close to the DPE30
. Proper function of a DPE containing promoter requires an Inr element, probably because these elements cooperatively promote the correct binding of TFIID30
. In summary, specific binding of the transcription machinery at the core promoter derives from cooperative binding of two or more general transcription factor subunits to degenerate, low specificity promoter elements. The combination of these elements varies between promoters and, in some cases, the core promoter elements used determine activator and enhancer specificity31
The role of TBP at TATA-less promoters.
It was initially believed that most Pol II promoters contained a TATA element, however, subsequent sequence analysis showed that only about 30% of mRNA genes analyzed in Drosophila
contain a recognizable TATA32
. Although TBP can recognize divergent AT-rich sequences because of its DNA binding mechanism (see below), there are a number of promoters which clearly do not have a TATA-like sequence about 30 bp upstream of the transcription start site that would be compatible with specific binding by TBP33
. In Drosophila
and human promoters, many of these non-TATA containing promoters contain some combination of Inr and DPE elements20
Must TBP bind DNA in order to function? At promoters with a functional TATA, mutation of TATA away from the consensus severely decreases transcription34
. From biochemical studies of the yeast HIS4 promoter, mutation of the TATA to a GC-rich sequence allowed recruitment of the transcription machinery to a promoter at a reduced level, but transcription initiation was completely abolished35
. These results demonstrate that at one class of promoter, assembly of the transcription machinery into a productive complex requires that TBP bind the TATA element as seen in the crystal structure. Although TBP has tremendous flexibility in the ability to bind variants of the TATA sequence, not all sequences are compatible with TBP binding. For example, C or G in certain positions within TATA is incompatible with the DNA binding surface of TBP33
. Since a large number of human and Drosophila
promoters have no recognizable TATA element or even AT-rich regions upstream from the transcription start32
, this suggests that if TBP interacts with DNA at these promoters, it must do so by a different mechanism from that seen at classical TATA elements. In support of this model, a mutation at the TBP binding surface that abolished detectable binding to a TATA element in vitro
blocked transcription from a TATA-containing promoter but not from an Inr-containing promoter36
. At promoters lacking TATA, TBP may nucleate protein-protein interactions among the general transcription factors and interact non-specifically with DNA, while DNA bending is facilitated by interaction of other factors such as TAFs with Inr and DPE elements.
Although there is only one gene encoding TBP in yeast and most archaea, higher eukaryotes have one or two copies of genes encoding TBP Related Factors (TRFs) in addition to TBP37,38
. It is well established that TRFs promote transcription from a subset of Pol II genes in a cell-type specific fashion. Trf1, unique to insect cells, binds a TC-rich sequence rather than a TATA element and promotes transcription from a small subset of Pol II promoters as well as all Drosophila
Pol III transcription39,40
. Trf2, conserved between Drosophila
, mouse, and human also directs transcription from a subset of promoters. Like TBP, Trf1 and Trf2 are both components of multi subunit complexes, although the identity of most of the Trf associated factors is not yet known.