**3. Characterization of ribosomal protein gene (RPG) promoters of** *Schizosaccharomyces pombe* **and their transcription initiation mechanism**

#### **3.1 The** *Schizosaccharomyces pombe* **RPG transcriptional module: the HomolD-box**

The characterization of the promoter sequences of 14 RPGs from the fission yeast *Schizosaccharomyces pombe* showed discrete conserved modules, which were

**155**

**Table 1.**

*Transcriptional Initiation in Ribosomal Protein Genes in the Fission Yeast…*

named Homol A, B, C, D, and E (**Table 1**) [36–38]. These homology regions were completely different from those described in promoters of genes from other yeasts and mammals, such as TATA-box, Inr, or DPEs. The function of each Homol element was studied using a promoter-deletion mutant approach [37]. This work showed that the role of Homol A, B, C, and E is associated to the regulation of transcription initiation, and that they might have a upstream activation sequence (UAS)-like function. Only the HomolD sequence was able to function as an element that could direct transcription initiation in the same way as the TATA-box [36]. The conserved sequence of the HomolD-box is the octamer CAGTCACA/G; however, in several sequences, this element is found in the inverted form as TGTGACTG. The HomolD-box is located 39–52 bp upstream of the transcription start site in the same position as the TATA-box in the fission yeast promoters. In an *in vivo* approach, using reporter-gene assays in *S. pombe* cells, it was shown that the HomolD-box is necessary to direct and initiate transcription from the RPG and was postulated to act as a TATA-box analog; in the same work, using an electrophoretic mobility shift assay (EMSA), a novel protein complex that binds to the HomolD-box was identified [36]. In other studies using an *in vitro* approach, it was shown that point mutations in the HomolD-box sequence abolish completely the ability of this element to

Currently, we know that the genome of *Schizosaccharomyces pombe* contains 141 RPGs encoding the full set of 79 ribosomal proteins. Interestingly, the analysis of the promoter sequences showed that 140 RPGs contained a highly conserved HomolDbox in the region 49–104 bp upstream of the ATG start codon [40]. Additionally, other 59 non-RPGs also showed the presence of the HomolD-box in their promoters. In addition, using promoter databases, it was possible to find HomolD-box sequences in several promoters from other eukaryotic organisms, such as humans and plants, indicating the broad distribution of this novel CPE. Moreover, a functional HomolDbox was found in the human ATPV1H gene where RECQL/DDB1 complex binds to

Interestingly, HomolD-boxes in RPG promoters are broadly distributed in the *Ascomycota* fungus phylum [42]. However, in those organisms closely related to the yeast *Saccharomyces cerevisiae,* other CPEs, in the same position as the HomolD-box, are present in RPG promoters. These elements are named Rap1 and bind the transcription factor Rap1p [43]. It seems likely that Rap1 replaced the HomolD-box of *Schizosaccharomyces pombe* in *Saccharomyces cerevisiae* during evolution. Moreover, several other yeast species share both HomolD-box and Rap1 promoter elements [42]. Taking all those observations together, we suggest that RPGs from *S. pombe*, *S. cerevisiae*, *Drosophila,* and mammals form a transcriptional module that is under the control of the HomolD-box, Rap1-box,

**Homol Consensus Binding TF Function Reference** HomolA TCAGTAACGAA Unknown UAS-like [48] HomolB AAAAGCTATG Unknown UAS-like [48] HomolC AAGAGTAAAATCT Unknown UAS-like [48]

> RECQL/DDB1 (Human, *S. pombe*)

HomolE AGGGTAGGGT Unknown UAS-like [37, 48]

Transcription initiation and regulation of RPG expression

[36, 39, 48]

*DOI: http://dx.doi.org/10.5772/intechopen.80602*

direct transcription initiation from the RPG [39].

this sequence and is required for *in vitro* transcription [41].

and TCT motif (*Drosophila* promoter element), respectively.

HomolD CAGTCACA/G Rrn7 (*S. pombe*)

*Homol sequences identified in RPG promoters in S. pombe.*

#### *Transcriptional Initiation in Ribosomal Protein Genes in the Fission Yeast… DOI: http://dx.doi.org/10.5772/intechopen.80602*

*Gene Expression and Control*

in most of them the TATA-box is absent. Different studies have determined that only 10–15% of mammal core promoters contain a TATA-box element [18–20]. Those promoters that do not contain a TATA-box were named TATA-less promoters and

After the identification of TATA-box sequence, other conserved promoter elements were identified. One of them is the initiator element (Inr), identified as a conserved DNA element in the region near to the transcription start site [21]. This element can not only direct transcription initiation by itself if other CPEs are not present, but also act synergistically in the presence of a TATA-box [22]. The proteins TAFII150 and TAFII250 have been identified as the transcription factors that are able to recognize the Inr and allow the formation of the PIC in Inr-containing promoters [23, 24]. However, other Inr-containing promoters might be able to direct transcription initiation in a TAFs-independent manner. In those promoters, a few proteins have been identified as Inr-binding factors, such as TFII-I and YY1 [25, 26]. Also, in other reports, transcription initiation from the human DNA beta polymerase promoter and from the human dihydrofolate reductase (DHFR) promoter, both TATA-less and Inr-containing promoters, has been achieved using solely TBP, IIB, IIE, IIF, IIH, and RNA pol II [26, 27]. This suggests that in some TATA-less promoters, the formation of a functional PIC might follow a common

Another CPE that has been described in TATA-less promoters is the downstream promoter element (DPE), identified first in *Drosophila melanogaster* [28]. This element is widely distributed in metazoan organisms and is located 28–32 bp downstream from the transcription start site and can be contained in the context of a TATA-box and/or an Inr. Studies in *Drosophila* have shown that proteins TAFII40 and TAFII60 might bind to the DPE to improve transcription initiation [29, 30]. Similar

Several other CPEs have been identified in TATA-less promoters but their contribution to transcription initiation is still poorly understood. Such is the case of motif ten element (MTE) [31]; TFIIB recognition element (BRE) [32]; X core promoter element 1 and 2 (XCPE1 and 2) [33, 34], both of which are able to direct transcription initiation; and the poly-pyrimidine initiator motif (TCT) motif [35]. The TCT

However, using the information from the sequencing of the genomes of other

organisms and the new bioinformatics technologies, it is expected that novel conserved CPEs will be identified and characterized and the transcription initiation mechanisms of TATA-less promoters will be revealed. Such is the case of the ribosomal protein genes (RPGs) in the fission yeast *Schizosaccharomyces pombe*, whose promoters do not contain a TATA-box; instead they possess a conserved sequence, acting as a TATA-analog to direct transcription initiation in those genes. In the next section, the RPG promoter of the fission yeast will be described and the transcrip-

**3. Characterization of ribosomal protein gene (RPG) promoters of** *Schizosaccharomyces pombe* **and their transcription initiation** 

**3.1 The** *Schizosaccharomyces pombe* **RPG transcriptional module: the** 

The characterization of the promoter sequences of 14 RPGs from the fission yeast *Schizosaccharomyces pombe* showed discrete conserved modules, which were

motif element will be described in another section of this chapter.

they have also been studied and different CPEs have been characterized.

pathway with those TATA-containing promoters.

elements have not been found in yeast yet.

tion initiation mechanism will be discussed.

**154**

**mechanism**

**HomolD-box**

named Homol A, B, C, D, and E (**Table 1**) [36–38]. These homology regions were completely different from those described in promoters of genes from other yeasts and mammals, such as TATA-box, Inr, or DPEs. The function of each Homol element was studied using a promoter-deletion mutant approach [37]. This work showed that the role of Homol A, B, C, and E is associated to the regulation of transcription initiation, and that they might have a upstream activation sequence (UAS)-like function. Only the HomolD sequence was able to function as an element that could direct transcription initiation in the same way as the TATA-box [36]. The conserved sequence of the HomolD-box is the octamer CAGTCACA/G; however, in several sequences, this element is found in the inverted form as TGTGACTG. The HomolD-box is located 39–52 bp upstream of the transcription start site in the same position as the TATA-box in the fission yeast promoters. In an *in vivo* approach, using reporter-gene assays in *S. pombe* cells, it was shown that the HomolD-box is necessary to direct and initiate transcription from the RPG and was postulated to act as a TATA-box analog; in the same work, using an electrophoretic mobility shift assay (EMSA), a novel protein complex that binds to the HomolD-box was identified [36]. In other studies using an *in vitro* approach, it was shown that point mutations in the HomolD-box sequence abolish completely the ability of this element to direct transcription initiation from the RPG [39].

Currently, we know that the genome of *Schizosaccharomyces pombe* contains 141 RPGs encoding the full set of 79 ribosomal proteins. Interestingly, the analysis of the promoter sequences showed that 140 RPGs contained a highly conserved HomolDbox in the region 49–104 bp upstream of the ATG start codon [40]. Additionally, other 59 non-RPGs also showed the presence of the HomolD-box in their promoters. In addition, using promoter databases, it was possible to find HomolD-box sequences in several promoters from other eukaryotic organisms, such as humans and plants, indicating the broad distribution of this novel CPE. Moreover, a functional HomolDbox was found in the human ATPV1H gene where RECQL/DDB1 complex binds to this sequence and is required for *in vitro* transcription [41].

Interestingly, HomolD-boxes in RPG promoters are broadly distributed in the *Ascomycota* fungus phylum [42]. However, in those organisms closely related to the yeast *Saccharomyces cerevisiae,* other CPEs, in the same position as the HomolD-box, are present in RPG promoters. These elements are named Rap1 and bind the transcription factor Rap1p [43]. It seems likely that Rap1 replaced the HomolD-box of *Schizosaccharomyces pombe* in *Saccharomyces cerevisiae* during evolution. Moreover, several other yeast species share both HomolD-box and Rap1 promoter elements [42]. Taking all those observations together, we suggest that RPGs from *S. pombe*, *S. cerevisiae*, *Drosophila,* and mammals form a transcriptional module that is under the control of the HomolD-box, Rap1-box, and TCT motif (*Drosophila* promoter element), respectively.


#### **Table 1.**

*Homol sequences identified in RPG promoters in S. pombe.*

#### **3.2 The role of Rrn7 and CK2 in RPG transcription initiation in**  *Schizosaccharomyces pombe*

The HomolD-box present in the RPG promoters of the fission yeast is the target of a DNA-binding protein with biochemical features different from TBP. The identification of the HomolD-box-binding protein was achieved using DNA affinity chromatography with double-stranded tandem HomolD-boxes covalently attached to a resin. Proteins bound to the resin were eluted and analyzed by mass spectrometry. The result was that the transcription factor Rrn7 was identified in the protein DNA-bound fraction [39]. This factor is a member of the RNA pol I transcriptional machinery and its function is to transcribe rDNA in the nucleolus. In the rDNA promoter, this factor is able to bind to a conserved box, which is similar to a HomolDbox. Rrn7 showed a specific HomolD-box-binding activity and it is required for the specific transcription of RPGs containing a HomolD-box [39]. Moreover, the GTFs and RNA pol II were required for accurate transcription initiation of a HomolDbox-containing promoter.

Rrn7 is part of the Zn-ribbon protein family related to TFIIB, including the mammalian ortholog TAF1B [44]. It possesses a Zn-ribbon domain in the N-terminal region and two cyclin-like domains in the carboxy-terminal region, displaying domain conservation with the TFIIB family members [44]. Recently, it has been demonstrated that *Schizosaccharomyces pombe* Rrn7 is able to interact with casein kinase 2 (CK2) both *in vitro* and *in vivo*, leading to a functional phosphorylation of threonine 67 in the N-terminal domain. This modification modulates negatively the transcriptional activity of Rrn7, affecting HomolD-directed transcription and DNA-binding activity [45]. Studies in *S. pombe* cell cultures using the specific CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) have shown the potentiation of RPG expression during CK2 inhibition. Moreover, using chromatin immunoprecipitation assays, it has been found that CK2 is associated with RPG promoters, suggesting that this kinase has a role in the modulation of ribosomal protein abundance [45].

#### **3.3 Preinitiation complex (PIC) formation on the RPG promoters in**  *Schizosaccharomyces pombe*

As stated before, RPGs that contain a HomolD-box are transcribed by the RNA pol II transcription apparatus [39]. The formation of the PIC on a HomolD-boxcontaining promoter was recently described [45, 46]. The first step in the formation of a PIC on these promoters is the binding of Rrn7 to the HomolD-box. As mentioned previously, this step in the PIC establishment might be regulated by phosphorylation of Rrn7 via CK2 protein kinase [45]. Upon the binding of Rrn7 to the HomolD-box, the general transcription factors TBP and TFIIB are able to recognize this DNAprotein complex [46]. After the binding of TBP/TFIIB to the complex, the RNA pol II/TFIIF complex is recruited, which in turn allows the TFIIE factor to be incorporated into the complex [46]. Finally, the mediator and the coactivator PC4 may be incorporated into the PIC and might modulate basal transcription through a putative HomolE-binding factor in those promoters that contain this DNA element. All the steps describing the pathway of complex formation are summarized in **Figure 1**.
