The general transcription factor TFIIB (transcription factor IIB) plays a critical role in the assembly of the RNA polymerase II pre-initiation complex. TFIIB can make sequence-specific DNA contacts both upstream and downstream of the TATA box. This has led to the definition of two core promoter BREs (TFIIB-recognition elements), one upstream [BREu (upstream BRE)] and one downstream of TATA box [BREd (downstream BRE)]. TFIIB–BREu and TFIIB–BREd contacts are mediated by two independent DNA-recognition motifs within the core domain of TFIIB. Both the BREu and the BREd modulate the transcriptional potency of a promoter. However, the net effect of the BREs on promoter activity is dependent on the specific blend of elements present within a core promoter.
Core promoters transcribed by RNA pol II (polymerase II)
The core promoter of genes transcribed by RNA pol II contains DNA sequence elements that are recognized by the general transcription machinery (reviewed in [1–3]). These elements play a crucial role in the regulation of transcription (reviewed in [4–6]). The first core promoter element to be characterized was the TATA box, which is recognized by the TBP (TATA-box–binding protein) subunit of TFIID (transcription factor IID). The TATA box acts as a positive regulatory element in transcription, providing a focal point and platform for the assembly of the PIC (pre-initiation complex) [7,8].
The Inr (initiator) element, which encompasses the transcription start site, is also able to nucleate PIC formation via components of TFIID. The Inr can function independently or in combination with a TATA element, contributing to the strength of TATA-containing promoter and accurate initiation (reviewed in ). The DPE (downstream promoter element) was identified at TATA-less promoters and its mutation results in a dramatic reduction in transcription . Like the Inr, the DPE is contacted by subunits of TFIID. A recently reported core promoter sequence, the MTF (motif ten element), can co-operate with the Inr to stimulate transcription . Here, we will describe two BREs (TFIIB-recognition elements) and discuss their roles in the regulation of transcription.
Transcription initiation requires assembly of general transcription factors at the promoter to form a PIC (reviewed in ). TFIIB plays a critical role in this process, providing a bridge between TFIID–DNA and pol II/TFIIF (reviewed in ). TFIIB is a single-chain polypeptide that is composed of an N-terminal zinc ribbon and a C-terminal domain, between which lies a highly conserved region termed the B-finger. The zinc ribbon is required to recruit the pol II/TFIIF to the PIC [13,14]. The C-terminal region of TFIIB binds to the TFIID–DNA complex with high affinity, interacting with the C-terminal stirrup of TBP .
The N- and C-terminal domains of TFIIB can engage in an intramolecular interaction that can be disrupted by the activation domain of VP16 (virion protein 16) . In addition, the B-finger region of TFIIB forms part of an important molecular switch, modulating the conformation of TFIIB (reviewed in ). The conformation of TFIIB probably modulates multiple functions, such as promoter recognition, the assembly of PIC, transcription start site selection and transcription activation [17–23].
TFIIB contacts the promoter both upstream and downstream of TATA box
Several previous studies had suggested that TFIIB could make extensive DNA contacts both upstream and downstream of TATA box of the promoter [24–26]. The crystal structure of TFIIBc–TBP2–TATA element ternary complex provided direct evidence that TFIIB binds to the DNA major groove immediately upstream of TATA element and the DNA minor groove immediately downstream of TATA element . However, this crystallographic structure did not provide a complete picture regarding the interaction between TFIIB and the DNA upstream and downstream of TATA element because a 16-bp DNA fragment with only 3 bp upstream of the TATA element was used in the resolved crystal structure. Using site-specific protein–DNA photocross-linking, the Ebright and Reinberg laboratories performed high-resolution mapping of TBP–TFIIA–TFIIB–promoter quaternary complex, demonstrating that TFIIB makes more extensive contacts with DNA upstream and downstream of TATA box than was previously shown and that the interaction between TFIIB and DNA upstream and downstream of TATA box may be highly asymmetrical .
Subsequent crystallographic analysis of TBPc–TFIIBc–DNA with an extended DNA fragment has confirmed the extensive contact that TFIIB can make with DNA both upstream and downstream of the TATA box at the AdMLP (adenovirus major late promoter) [29,30]. In addition, our previous work had suggested that TFIIB makes sequence-specific DNA contacts downstream of TATA box in adenovirus E4 promoter . Taken together, these works suggest that the TFIIB-recognition regions upstream and downstream of TATA box might contain core promoter elements.
BREs modulate promoter strength
The first defined BRE [later termed BREu (upstream BRE)] is located immediately upstream of TATA box. The BREu consensus sequence is 5′-G/C-G/C-G/A-C-G-C-C-3′ (Figure 1) . In vitro experiments using purified basal transcriptional factors demonstrated that the interaction between TFIIB and BRE enhances the assembly of TBP–TFIIB–DNA and transcription initiation . Contact with the BRE is mediated by an HTH (helix–turn–helix) motif located at the C-terminus of TFIIB that binds through the major groove of the DNA. The TFIIB–BRE contact is conserved in Archaea, although the sequences of the BRE are not similar . Interestingly, however, TFIIB–BRE contact is not conserved in yeast and plants .
A scheme of the known core promoter elements, showing the locations of the BREu and the BREd relative to the TATA box, the Inr (INR), MTE and DPE
The BRE can also act as a negative element in transcription [33,34]. The net effect of this is to increase the amplitude of transcription activation in the presence of Gal4-VP16. Interestingly, the activation domain of VP16 can disrupt the TFIIB–BRE interaction within a promoter-bound complex . The disruption to TFIIB–BRE interaction by VP16 may therefore contribute to the process of transcription stimulation. However, the mechanism involved in this event remains to be investigated. Thus the BREu may act as positive or negative element in transcriptional regulation.
In a recent study, we have defined the core promoter element downstream of TATA box that is recognized by TFIIB [termed BREd (downstream BRE)] . The consensus sequence of BREd is 5′-G/A-T-T/G/A-T/G-G/T-T/G-T/G-3′ (Figure 1). The BREd modulates promoter strength (in both basal and activator-dependent transcription), acting as a positive element in the assembly of TBP–TFIIB-promoter complex. Interestingly, the BREd regulates transcription in a promoter context-dependent manner. The BREd elicits a positive effect on transcription when the promoter contains only a BREd. However, the BREd has a negative effect on transcription when the promoter also bears a BREu. It is therefore likely that the BREs are interdependent in their effects on core promoter strength.
The modes for TFIIB binding to the BREu and BREd
In a crystal structure of TFIIBc–TBP2–DNA ternary complex, TBP2 binds the TATA element of the AdMLP, causing severe bending of the DNA. TFIIB then acts as a clamp, binding to the cleft of TBP2 and interacting with the negatively charged phosphodiester backbone of the DNA upstream and downstream of TATA box rather than the edges of bases of the DNA. Therefore TFIIB incorporation can increase the affinity of TBP for the core promoter, stabilizing the TFIIB–TBP–DNA complex .
The HTH motif of TFIIB is located within the second cyclin-like repeat of TFIIB (residues Thr270–Arg290), consisting of helices BH4′ and BH5′. In the interaction between BH5′ and BREu, the residue Val283 interacts with the C5-C6 edge of base C(–34), 3 bp upstream of the TATA box . The base pair (G:C) at –34, one of important signatures of the BREu, is conserved in 36% of all human and 56% of viral pol II promoters . The specificity of Val283 for the G:C base-pair at –34 of the BREu was confirmed by the mutation of Val283 (V283A), which results in a significant reduction in transcription and assembly of TIIB–TBP–DNA . In addition, the side chain of Arg286 makes water-mediated contact with the edges of bases G(–38) and G(–37), and interacts with the phosphoribose backbone of G (–38) . The mutation of Arg286 also reduces transcription in vitro, caused by reduced assembly of TFIIB–TBP–DNA complex . The sequence of the HTH in TFIIB is conserved in all eukaryotes, especially several residues at the recognition surface between TFIIB and G:C base-pairs at positions –36 and –37 . However, as mentioned previously, the HTH motif is not conserved in plant and yeast TFIIBs, implying that TFIIB interacts with the BREu differently in these organisms [29,31].
TFIIB contacts the DNA minor groove downstream of the TATA box (BREd) through a loop (residues 152–156 of human TFIIB) between BH2 and BH3 in the first cyclin-like repeat of TFIIB [29,35]. In the interaction between the loop and BREd, Gly153 makes a base-specific contact with the 2-amino group of the G at –20, Arg154 makes a water-mediated contact with the 3-amino group of the G at –19 of the AdMLP . The specificities of Gly153 and Arg154 for the BREd have been confirmed by the mutation of these residues, which caused a severe reduction in transcription and assembly of TBP–TFIIB–DNA . In addition, the side chains of several residues (Lys152, Arg154, Ala155 and Asn156) within the loop make van der Waals or polar contacts with the DNA minor groove downstream of the TATA box . This would strengthen the interaction between the recognition loop and the BREd. Interestingly, unlike the HTH motif in TFIIB, the sequence of the loop is highly conserved in all eukaryotes and archaea, especially Gly153 and Arg154. In summary, TFIIB has evolved two independent DNA-recognition motifs: the HTH in the C-terminal cyclin-like repeat of TFIIB, contacting the BREu, and the loop between BH2 and BH3 in the N-terminal cyclin-like repeat of TFIIB, which recognizes the BREd.
The prevalence of the BREs
Several core promoter elements have now been described. None appear to be universal. Instead, each core promoter contains a subset of elements. A recent bioinformatics study revealed that nearly half of promoters (49%) have the Inr element at a functional position, 21.8% contain TATA elements and 24.6% have a DPE . This same study showed that 25% of core promoters from the EPD (Eukaryotic Promoter Database; http://www.epd.isb-sib.ch/index.html) have BREu. The TATA-less promoters are more likely to contain a BREu than the TATA-containing promoters (28.1% versus 11.8%). Thus it is possible that BREs might, at least in some cases, functionally substitute for the TATA box in nucleating the formation of a TFIIB–TBP–promoter complex. We have found that the BREd has prevalence similar to that of the BREu . Interestingly, our preliminary analysis suggests that the BREs show an independent distribution among core promoters. However, it remains to be determined if this generally applies when other specific core promoters are taken into consideration. It is also very likely that additional core promoter elements are yet to be identified. Further studies will therefore be required to determine the relationships between core promoter elements and their roles in determining transcription potency.
Molecular Basis of Transcription: A Focus Topic at BioScience2006, held at SECC Glasgow, U.K., 23–27 July 2006. Edited by S. Busby (Birmingham, U.K.), R. Weinzierl (Imperial College London, U.K.) and R. White (Glasgow, U.K.).
adenovirus major late promoter
transcription factor IIB
downstream promoter element
motif ten element
- pol II
virion protein 16
We thank the Wellcome Trust for support. S.G.E.R. is a Wellcome Trust Senior Fellow.