**4. Regulation of RPG expression in** *Schizosaccharomyces pombe*

The expression of genes containing the HomolD-box in their promoters is almost unknown. However, data from analysis of the RPG expression profiles during

**157**

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

several biological processes in *S. pombe*, for example, the switch from vegetative to meiotic growth and growth under stress conditions, have revealed a tightly coordinated expression for all 141 RPGs. For example, during the switch from vegetative to meiotic growth, transcription of RPG is down-regulated, but then, within a short time, strong reactivation of RPG expression is observed at the beginning of meiosis. The same co-regulation profile is observed in 32 of the 59 non-RPGs that contain a HomolD-box in their core promoter [40]. Many, but not all, of these non-RPGs encode components whose homologs in other organisms are involved in protein

Several promoters of *Schizosaccharomyces pombe* RPGs have been isolated and characterized [47–50], showing that individual ribosomal proteins are encoded by two or three related genes whose promoters contain a HomolD-box. Interestingly, in each gene family, at least one promoter possesses a tandem repeat ACCCTACCCT or the inverted form (AGGGTAGGGT) upstream of the HomolD-box. This sequence corresponds to the HomolE-box, which is considered a proximal UAS-like sequence for HomolD-box-containing promoters, since the presence of this element strongly increases *in vivo* transcription directed by the HomolD-box. Both promoter elements HomolD- and HomolE-boxes must be in the same orientation to be functional. The distance between the boxes is critical in transcription modulation of RPGs, and it has been described that the smaller the distance between HomolD and HomolE, the higher the transcription activity. This distance ranges from 0 to 32 nucleotides.

Now that the complete genome of *Schizosaccharomyces pombe* is available and searchable, it is known that of the 141 RPG promoters, 140 promoters contain a HomolD-box and 62 contain a HomolE-box upstream of the HomolD-box. In contrast, only 5 of the 59 non-RPG promoters containing a HomolD-box contain a HomolE-box [40].

The activity of the HomolE-box must be related to a transcriptional activator in the same manner as the IFH1 element is recognized by a TF (Ifh1p) in *Saccharomyces cerevisiae* RPGs [51, 52]. This element has been identified upstream of the Rap1 sequence in RPG promoters. It is likely that a member of the same family of Ifh1p recognizes the HomolE-box in *S. pombe*. However, the gene encoding this protein

The modular architecture of the *Schizosaccharomyces pombe* RPG promoters where the HomolE-box is always found upstream of HomolD indicates that some of the promoters are under the control of the HomolE-box. This fact suggests that there must be a mechanism that regulates RPG transcription through the HomolE-

Further investigations must be performed to understand how RPG expression is regulated and which are the mechanisms involved in the coordination between HomolD- and HomolE-boxes during RPG transcription. Despite the fact that there are several factors and mechanisms studied in RNA pol II-directed transcription, most of the promoters studied possess a TATA-box, whereas RPG promoters are TATA-less. Moreover, transcriptional initiation and activation from TATA-less promoters are poorly understood both in metazoan and yeast cells. Thus, the RPG promoters and the arrangement of HomolE-HomolD could provide a model to study transcription in TATA-less promoters using a promoter element such as

**5. Coordinated regulation of the expression of ribosomal components in** 

Ribosome biogenesis is one of the most complicated processes in eukaryotic cells, requiring coordinated expression of all ribosome components, which are

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

biosynthesis and signal transduction [40].

has not been identified yet.

box under specific growth conditions.

HomolD that is analogous to the TATA-box.

*Schizosaccharomyces pombe*

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

*Gene Expression and Control*

box-containing promoter.

protein abundance [45].

*Schizosaccharomyces pombe*

*Schizosaccharomyces pombe*

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

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 HomolD-

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

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

**4. Regulation of RPG expression in** *Schizosaccharomyces pombe*

unknown. However, data from analysis of the RPG expression profiles during

The expression of genes containing the HomolD-box in their promoters is almost

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**.

**156**

several biological processes in *S. pombe*, for example, the switch from vegetative to meiotic growth and growth under stress conditions, have revealed a tightly coordinated expression for all 141 RPGs. For example, during the switch from vegetative to meiotic growth, transcription of RPG is down-regulated, but then, within a short time, strong reactivation of RPG expression is observed at the beginning of meiosis. The same co-regulation profile is observed in 32 of the 59 non-RPGs that contain a HomolD-box in their core promoter [40]. Many, but not all, of these non-RPGs encode components whose homologs in other organisms are involved in protein biosynthesis and signal transduction [40].

Several promoters of *Schizosaccharomyces pombe* RPGs have been isolated and characterized [47–50], showing that individual ribosomal proteins are encoded by two or three related genes whose promoters contain a HomolD-box. Interestingly, in each gene family, at least one promoter possesses a tandem repeat ACCCTACCCT or the inverted form (AGGGTAGGGT) upstream of the HomolD-box. This sequence corresponds to the HomolE-box, which is considered a proximal UAS-like sequence for HomolD-box-containing promoters, since the presence of this element strongly increases *in vivo* transcription directed by the HomolD-box. Both promoter elements HomolD- and HomolE-boxes must be in the same orientation to be functional. The distance between the boxes is critical in transcription modulation of RPGs, and it has been described that the smaller the distance between HomolD and HomolE, the higher the transcription activity. This distance ranges from 0 to 32 nucleotides.

Now that the complete genome of *Schizosaccharomyces pombe* is available and searchable, it is known that of the 141 RPG promoters, 140 promoters contain a HomolD-box and 62 contain a HomolE-box upstream of the HomolD-box. In contrast, only 5 of the 59 non-RPG promoters containing a HomolD-box contain a HomolE-box [40].

The activity of the HomolE-box must be related to a transcriptional activator in the same manner as the IFH1 element is recognized by a TF (Ifh1p) in *Saccharomyces cerevisiae* RPGs [51, 52]. This element has been identified upstream of the Rap1 sequence in RPG promoters. It is likely that a member of the same family of Ifh1p recognizes the HomolE-box in *S. pombe*. However, the gene encoding this protein has not been identified yet.

The modular architecture of the *Schizosaccharomyces pombe* RPG promoters where the HomolE-box is always found upstream of HomolD indicates that some of the promoters are under the control of the HomolE-box. This fact suggests that there must be a mechanism that regulates RPG transcription through the HomolEbox under specific growth conditions.

Further investigations must be performed to understand how RPG expression is regulated and which are the mechanisms involved in the coordination between HomolD- and HomolE-boxes during RPG transcription. Despite the fact that there are several factors and mechanisms studied in RNA pol II-directed transcription, most of the promoters studied possess a TATA-box, whereas RPG promoters are TATA-less. Moreover, transcriptional initiation and activation from TATA-less promoters are poorly understood both in metazoan and yeast cells. Thus, the RPG promoters and the arrangement of HomolE-HomolD could provide a model to study transcription in TATA-less promoters using a promoter element such as HomolD that is analogous to the TATA-box.
