Starting from a genome-wide annotation of TFBSs for
yeast regulatory motifs, we comprehensively studied TFBS positioning and nucleosome coverage profiles across Saccharomyces cerevisiae
promoters. We uncovered that TATA-box containing and TATA-less promoters have significantly different architectures. Compared to TATA-less promoters TATA promoters show an overall lower number of TFBSs per promoter, these TFBSs occur further upstream of TSS on average, and show a wider distribution of distances with respect to TSS. We find that the TFBS profiles closely mirror nucleosome coverage profiles, i.e. TATA promoters have higher nucleosome coverage, the region of lowest nucleosome coverage occurs further upstream, and the region of lowest nucleosome coverage is more sharply defined in TATA-less promoters.
There recently has been a large amount of investigation into the mechanisms that determine nucleosome positioning, and the extent to which nucleosome positioning is determined by intrinsic sequence preferences of the nucleosomes is currently actively disputed, see 
. Since nucleosome positioning is not the main topic of this work, we do not wish to enter into this debate here. However, we do note that the remarkably close and consistent match that we observed between TFBS density profiles and nucleosome coverage profiles across different subsets of promoters, strongly suggests that competition between TFs and nucleosomes for binding to DNA likely plays an substantial role in shaping nucleosome occupancy profiles in yeast promoters.
Whereas the position of overall highest TFBS density occurs more than
bps upstream of TSS, the TATA motif itself has highest density more proximal to TSS, i.e. at approximately
bps upstream of the TSS. This is still considerably further upstream than the location of the TATA-box in mammals, where it occurs about
bp upstream of TSS, and there is considerable evidence 
that, in yeast, the PIC is recruited significantly upstream of TSS and then ‘scans’ down the upstream sequence until it encounters the site where it initiates transcription. To investigate this scanning process we constructed an initiator motif, i.e. representing the sequences at the initiation site, and established that it is essentially identical in TATA and TATA-less promoters. Moreover, in TATA promoters the initiator motif has a maximum both at the TSS and at the position of highest density of TATA sites, suggesting that the PIC may initially be recruited to the position of the TATA sites, and start its scanning from this position. We also saw that in TATA-less promoters this peak in affinity of the initiator motif occurs closer to the TSS.
A key result of this study is that, besides the TATA motif, there are an additional
regulatory motifs that also preferentially occur proximal to TSS, i.e. between
bps upstream of TSS. These alternative proximal promoter motifs occur preferentially in TATA-less promoters and their positioning proximal to TSS is observed predominantly in TATA-less promoters. Moreover, the position of highest density of alternative PPMs in TATA-less promoters corresponds to the position at which the second maximum in initiator affinity occurs, suggesting that, just as the PIC is initially recruited to the TATA site in TATA promoters, in TATA-less promoters the PIC may be initially recruited to alternative PPMs. In addition, we showed that TATA-less promoters can be classified based on the PPM they contain, and that different classes of TATA-less promoters show distinct TFBS and nucleosome coverage distributions. provides a diagrammatic summary of the differences in architecture of TATA and TATA-less promoters identified in this study.
Diagrammatic summary of the architecture of TATA (left panel) and TATA-less (right panel) promoters.
Our results establish that many of the TATA-less promoters are characterized by the occurrence of alternative PPMs, and suggest that these play a crucial role in regulating transcription at these TATA-less promoters. The main question that now arises is what the precise functional role of these alternative PPMs is, and how their function relates to that of the TATA-box. With respect to the latter, although only about
of promoters contain a TATA-box, the TATA binding protein SPT15 is recruited to all promoters, and is generally required for transcription 
. Although the precise mechanism of function of the TATA site remains elusive, it is clear that at TATA promoters the TATA site is required for proper transcription 
, and one could imagine that the TATA site is requirement for recruitment of the PIC.
The simplest hypothesis for the functioning of the alternative PPMs, which is consistent with all our results, is to assume that they ‘replace’ the TATA site in TATA-less promoters, i.e. that these PPM sites are directly involved in recruiting the PIC. However, a review of the literature on the PPM motifs is at odds with this simple interpretation.
First, GATA sites are generally found upstream of genes that are subject to nitrogen catabolite repression 
. Very roughly, in nitrogen-rich media these sites are bound by the repressors DAL80/GZF3 while in nitrogen-poor media the sites are bound by GAT1 and GLN3, activating their target genes. Moreover, the signaling of nitrogen availability is mediated by the TOR1 complex 
with both GLN3 and GZF3 interacting directly with Tor1p 
. In addition, there is a significant amount of cross-regulation between the GATA factors themselves, including binding to each other's promoters 
. Thus, activator and repressor GATA TFs compete for binding to the GATA sites, and depending on nitrogen availability this competition will favor either the activating or repressing factors. Thus, although there is a reported case in the literature of a GATA site being recognized by TATA binding protein 
, the main function of the motif appears to be in mediating either repression of activation of genes in response to nitrogen levels.
The NDT80 motif is another example of a motif where a repressor and an activator TF compete for binding to target sites. NDT80 is a meiosis-specific TF that is required for exit from pachytene and that activates middle sporulation genes. During mitosis and in the vegetative state the same binding sites, which are also called middle sporulation elements (MSEs), are bound by the repressor SUM1, i.e. SUM1 acts as a brake on meiosis 
. Thus, as for the GATA motif, in nutrient-rich conditions the target sites are bound by a repressor, whereas under starvation, when the cells go into sporulation, the repressor is replaced by an activating TF.
A similar function applies to the third proximal promoter motif, ROX1. ROX1 is a heme-dependent repressor of hypoxic genes, i.e. under aerobic conditions ROX1 binds to its target sites while under anaerobic conditions its targets are derepressed 
. However, the fact that ROX1 is associated mostly with TATA promoters makes it a somewhat special case.
Fourth, the forkhead transcription factors FKH1 and FKH2 are key regulators of the cell-cycle in yeast, targeting the CLB2 cluster of genes which includes the downstream TFs SWI5 and ACE2 
. The two forkhead factors often compete for binding to the same promoters 
and interact with different chromatin remodeling complexes to repress target genes during the G2/M and G1 phases of the cell cycle 
. Thus, like in the previous examples, the forkhead TFs can act as repressors on their targets, effectively implementing a check-point that is released when they are displaced from their target sites.
Fifth, the PAC (Polymerase A and C) motif is found in the promoters of ribosome biogenesis and rRNA genes and it has recently been shown to be bound by the TFs PBF1 and PBF2 
. It has become clear that both PBF1 and PBF2 act as repressors on their targets genes and are activated upon stresses such as heat shock or nutrient signals, with the two TFs being responsive to different stress signals 
. It is as of yet not clear whether any other (activating) TF may bind to PAC sites under nutrient-rich conditions. The PBF motif thus seems to implement a similar check-point on nutrient availability, releasing its target ribosome biogenesis genes from repression when sufficient nutrients are available.
Finally, RPN4 is an activator of 26S proteasome genes which is itself rapidly degraded by the proteasome, generating a negative feed-back loop that controls proteasome homeostasis 
. RPN4 expression is controlled by stress responses and the feed-back loop between RPN4 expression and the proteasome is important for cell viability under various stresses 
. Thus in contrast to all other examples which involved binding by either repressors or competition between repressing and activating TFs for binding to the PPM, the RPN4 sites seem to be mainly targeted by the activator RPN4. However, RPN4 clearly plays a role in response to various stresses.
In summary, it appears that all PPMs are involved in responding to environmental stresses, often involving nutrient availability, either releasing (GATA, NDT80, ROX1) their target genes in response to the stress or (PBF, FKH) repressing their targets when stresses are present. Another feature shared by the PPMs is that, through competition of both activating and repressing TFs binding to the site, the PPM sites are essentially always bound. These features are consistent with their preferred targeting of TATA-less promoters.
Previous studies have shown that TATA promoters in yeast are characterized by closed chromatin, regulation through chromatin, and that many of the associated genes are upregulated upon various stresses. In contrast, ‘house-keeping’ genes tend to have TATA-less promoters 
. Simplifying one might say that, under nutrient-rich conditions, TATA promoters are ‘off’ by default and the TATA boxes are occluded by nucleosomes. In contrast, many of the TATA-less promoters are expressed and have a distinct nucleosome free region proximal to the TSS 
. Upon the appearance of various stresses many of the TATA promoters are induced whereas many of the TATA-less promoters are repressed. The alternative PPMs identified in this study appear to generally be involved in this switching in response to nutrient availability and other stresses. Our results suggest that, whereas TATA promoters may respond to a large diversity of stresses, the alternative PPMs may be involved with responding to specific stresses such as cell-cycle check points (FKH), nitrogen and carbon levels (GATA, NDT80, PBF), oxygen levels (ROX1), and heat shock (PBF). Whereas TATA sites may be occluded by nucleosomes in nutrient-rich conditions, most of the PPM sites switch between accommodating repressing and activating TFs, and are thus generally associated with regions depleted of nucleosomes.