**2. General features of transcription initiation in the eukaryotic organisms and TATA-less CPEs**

Transcription in eukaryotic organisms is carried out by RNA polymerases (RNA pols), which are enzymatic complexes composed by at least 12 subunits. In general, eukaryotic genes are classified as class I, II, and III, where class I genes codify rRNAs; class II codify pre-mRNAs; and class III codify 5S rRNA, tRNAs, and snRNAs, respectively. Transcription of each class of genes is carried out by a different RNA pol. Class I genes are transcribed by RNA pol I, class II genes are transcribed by RNA pol II, and class III genes are transcribed by RNA pol III, respectively. This specific transcription is based on the recognition of specific DNA sequences in the promoters of each class of genes by different transcription factors (TFs) that are able to recruit each specific RNA pol. These sequences are named "core promoter elements" (CPEs) and are located inside the region of the promoter named "core promoter" (CP) that is able to direct the formation of a pre-initiation complex (PIC) and initiate specific transcription of the gene. The CPEs are recognized by TFs specific to each RNA pol, which are called "general transcription factors" (GTFs). In summary, each RNA pol has a set of specific GTFs and these protein factors are able to recognize the CPEs associated to each class' gene promoters.

RNA pol II has been widely studied due to the enzyme that transcribes proteincoding genes. One of the first CPEs described in the promoters of class II genes was the so-called TATA-box [3–5]. This CPE is distributed in the promoters of most eukaryotic organisms and is located 25–40 bp upstream from the transcription initiation site. The formation of a PIC on the promoters containing a TATA-box has been extensively studied and characterized [6–8]. The formation of a PIC on the TATA-box starts with the recognition and binding of the transcription factor TATAbinding protein (TBP) to the TATA-box which in turn recruits the other GTFs and RNA pol II to form the PIC, which is able to initiate transcription upon the addition of the ribonucleotides [9, 10] (**Figure 1**). As it can be seen from the model, RNA pol II is integrated into the PIC in association with TFIIF when the promoter-TBP-TFIIB complex is formed. On the other hand, a fraction of RNA pol II can be purified from cell extracts in association with TFIIF and the mediator, and since those complexes are preformed inside the nucleus, a fast recruitment of the PIC to the promoter could be produced [11–13]. The multi-subunit complex named mediator (a general transcriptional coactivator) is also necessary for the transcription *in vivo*, in crude cell extracts, of class II genes [14]. In addition, recently, it has been demonstrated that another protein complex is recruited *in vivo* at most of the class II gene promotes in *S. cerevisiae*, where it plays a fundamental role in transcription. This multi-protein complex is named SAGA and is composed of several subunits including Gcn5, which have histone acetyltransferase (HAT) activity Spt gene products and TBP-associated factors (TAFs) that are shared with the complex TFIID [15].

In metazoan cells, the transcription factor TBP is tightly associated to TAFs and the TBP-TAF complex is named TFIID [16]. The role of TAFs seems to be the recognition of certain CPEs such as the Inr, motif ten element (MTE), and downstream promoter element (DPE) (see below). However, in yeast, this complex seems to be unstable, since it is possible to purify TBP free of TAFs from yeast cell extracts. Although TAFs are required for *in vivo* transcription of *S. cerevisiae* genes, the exact

**153**

**Figure 1.**

transcribe TATA-less promoters [17].

*box-containing promoters.*

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

mechanism of their function has not been identified yet. Interestingly, using an *in vitro* approach using TFIID-depleted yeast cell extracts, it was found that transcription from both TATA-containing and TATA-less promoters is dependent on TFIID, but isolated recombinant TBP can only rescue the transcription of TATA-containing promoters, indicating that additional interactions are necessary to efficiently

PIC formation on TATA-containing and HomolD-containing promoters**.** *Classical PIC formation on a TATA-containing promoter is outlined in A–F. First, TBP binds to the TATA-box and then TFIIB is recruited to the promoter-TBP complex. This allows the RNA pol II-TFIIF complex to be incorporated into the promoter-TBP-TFIIB complex (C). Once that RNA pol II-TFIIF is loaded onto the complex, the transcription factor TFIIE is incorporated (D) followed by the binding of TFIIH (E). The mediator complex might be incorporated into the complex after the binding of RNA pol II-TFIIF and TFIIE (D). At step F, the complete PIC is formed and it is competent for transcription initiation. A competent PIC is formed on HomolD-box containing promoters, such as RPG promoters, to initiate RNA pol II-dependent transcription (G–L). The first step is the binding of the transcription factor Rrn7 to the HomolD-box sequence (G). Then, transcription factors TBP and TFIIB bind to Rrn7 (H). This DNA-protein complex is recognized by RNA pol II-TFIIF (I) and TFIIE (J). This complex is competent to initiate HomolD-box-dependent transcription. However, coactivators such as the mediator, PC4, and the HomolE-binding factor would be necessary to modulate transcription initiation (K and L). TSS: transcription start site. Note that steps C to F for PIC formation on TATA-box-containing promoters might be common with steps I to L on PIC formation on HomolD-*

However, our vision of transcription initiation on TATA-box-containing promoters cannot explain the mechanisms of transcription initiation on all the class II genes, because the analysis of several other class II gene promoter sequences showed that

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

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

#### **Figure 1.**

*Gene Expression and Control*

the fission yeast *Schizosaccharomyces pombe*.

**organisms and TATA-less CPEs**

recognize the CPEs associated to each class' gene promoters.

factors (TAFs) that are shared with the complex TFIID [15].

module (regulon). In this chapter, the authors will describe the state of the art of several topics associated to the transcription initiation from TATA-less promoters in eukaryotic organisms, such as the transcriptional regulation of RPGs in metazoan cells and the description of a novel mechanism of regulation present in the RPG of

Transcription in eukaryotic organisms is carried out by RNA polymerases (RNA pols), which are enzymatic complexes composed by at least 12 subunits. In general, eukaryotic genes are classified as class I, II, and III, where class I genes codify rRNAs; class II codify pre-mRNAs; and class III codify 5S rRNA, tRNAs, and snRNAs, respectively. Transcription of each class of genes is carried out by a different RNA pol. Class I genes are transcribed by RNA pol I, class II genes are transcribed by RNA pol II, and class III genes are transcribed by RNA pol III, respectively. This specific transcription is based on the recognition of specific DNA sequences in the promoters of each class of genes by different transcription factors (TFs) that are able to recruit each specific RNA pol. These sequences are named "core promoter elements" (CPEs) and are located inside the region of the promoter named "core promoter" (CP) that is able to direct the formation of a pre-initiation complex (PIC) and initiate specific transcription of the gene. The CPEs are recognized by TFs specific to each RNA pol, which are called "general transcription factors" (GTFs). In summary, each RNA pol has a set of specific GTFs and these protein factors are able to

RNA pol II has been widely studied due to the enzyme that transcribes proteincoding genes. One of the first CPEs described in the promoters of class II genes was the so-called TATA-box [3–5]. This CPE is distributed in the promoters of most eukaryotic organisms and is located 25–40 bp upstream from the transcription initiation site. The formation of a PIC on the promoters containing a TATA-box has been extensively studied and characterized [6–8]. The formation of a PIC on the TATA-box starts with the recognition and binding of the transcription factor TATAbinding protein (TBP) to the TATA-box which in turn recruits the other GTFs and RNA pol II to form the PIC, which is able to initiate transcription upon the addition of the ribonucleotides [9, 10] (**Figure 1**). As it can be seen from the model, RNA pol II is integrated into the PIC in association with TFIIF when the promoter-TBP-TFIIB complex is formed. On the other hand, a fraction of RNA pol II can be purified from cell extracts in association with TFIIF and the mediator, and since those complexes are preformed inside the nucleus, a fast recruitment of the PIC to the promoter could be produced [11–13]. The multi-subunit complex named mediator (a general transcriptional coactivator) is also necessary for the transcription *in vivo*, in crude cell extracts, of class II genes [14]. In addition, recently, it has been demonstrated that another protein complex is recruited *in vivo* at most of the class II gene promotes in *S. cerevisiae*, where it plays a fundamental role in transcription. This multi-protein complex is named SAGA and is composed of several subunits including Gcn5, which have histone acetyltransferase (HAT) activity Spt gene products and TBP-associated

In metazoan cells, the transcription factor TBP is tightly associated to TAFs and the TBP-TAF complex is named TFIID [16]. The role of TAFs seems to be the recognition of certain CPEs such as the Inr, motif ten element (MTE), and downstream promoter element (DPE) (see below). However, in yeast, this complex seems to be unstable, since it is possible to purify TBP free of TAFs from yeast cell extracts. Although TAFs are required for *in vivo* transcription of *S. cerevisiae* genes, the exact

**2. General features of transcription initiation in the eukaryotic** 

**152**

PIC formation on TATA-containing and HomolD-containing promoters**.** *Classical PIC formation on a TATA-containing promoter is outlined in A–F. First, TBP binds to the TATA-box and then TFIIB is recruited to the promoter-TBP complex. This allows the RNA pol II-TFIIF complex to be incorporated into the promoter-TBP-TFIIB complex (C). Once that RNA pol II-TFIIF is loaded onto the complex, the transcription factor TFIIE is incorporated (D) followed by the binding of TFIIH (E). The mediator complex might be incorporated into the complex after the binding of RNA pol II-TFIIF and TFIIE (D). At step F, the complete PIC is formed and it is competent for transcription initiation. A competent PIC is formed on HomolD-box containing promoters, such as RPG promoters, to initiate RNA pol II-dependent transcription (G–L). The first step is the binding of the transcription factor Rrn7 to the HomolD-box sequence (G). Then, transcription factors TBP and TFIIB bind to Rrn7 (H). This DNA-protein complex is recognized by RNA pol II-TFIIF (I) and TFIIE (J). This complex is competent to initiate HomolD-box-dependent transcription. However, coactivators such as the mediator, PC4, and the HomolE-binding factor would be necessary to modulate transcription initiation (K and L). TSS: transcription start site. Note that steps C to F for PIC formation on TATA-box-containing promoters might be common with steps I to L on PIC formation on HomolDbox-containing promoters.*

mechanism of their function has not been identified yet. Interestingly, using an *in vitro* approach using TFIID-depleted yeast cell extracts, it was found that transcription from both TATA-containing and TATA-less promoters is dependent on TFIID, but isolated recombinant TBP can only rescue the transcription of TATA-containing promoters, indicating that additional interactions are necessary to efficiently transcribe TATA-less promoters [17].

However, our vision of transcription initiation on TATA-box-containing promoters cannot explain the mechanisms of transcription initiation on all the class II genes, because the analysis of several other class II gene promoter sequences showed that

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

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 pathway with those TATA-containing promoters.

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 elements have not been found in yeast yet.

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

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 transcription initiation mechanism will be discussed.
