Homol sequences identified in RPG promoters in
Transcription of class II genes in eukaryotic organisms is carried out by the multi-subunit enzyme RNA polymerase II (RNA pol II) and includes the general transcription factors and the mediator. The region inside the promoters, which recruits and specifies the transcriptional machinery, is called “core promoter” and contains sub-regions called “core promoter elements,” which are necessary for transcription initiation, where the most studied and classic element is the TATA-box. Ribosome protein gene (RPG) promoters do not possess a TATA-box (TATA-less promoters), and those, in particular, in the fission yeast Schizosaccharomyces pombe have a TATA-box analog called the HomolD-box. The transcription of RPG promoters is dependent on the RNA pol II transcription system and the HomolD-box is recognized by the transcription factor Rrn7. In this chapter, the authors will describe the general mechanisms associated to the transcription of TATA-less promoters in eukaryotic organisms and how the transcription initiation is carried out in the RPG promoters from those organisms, particularly in Schizosaccharomyces pombe. Finally, the authors will analyze the role of the HomolD-box and the transcription factor Rrn7 in the coordination of transcription initiation from RPG promoters and other ribosome-related genes and the presence of transcriptional modules in their promoters, which could be coordinated and regulated by a discrete number of transcription factors.
- ribosomal protein gene (RPG)
- RNA polymerase
- TATA-less promoter
- Schizosaccharomyces pombe
Protein synthesis in eukaryotic organisms includes several steps and requires many regulatory events [1, 2]. One of these critical steps is ribosome biogenesis, which includes ribosomal protein gene (RPG) transcription and rRNA synthesis. As in many other central events in the cell, ribosome biosynthesis must be a regulated and coordinated process. A typical coordinated regulation of gene and protein expression is the presence of common DNA elements in the promoters of related genes, which are co-regulated by a discrete number of transcription factors. Those genes under the control of a common DNA element form a transcriptional 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 the fission yeast
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 protein-coding genes. One of the first CPEs described in the promoters of class II genes was the so-called TATA-box [3, 4, 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, 7, 8]. The formation of a PIC on the TATA-box starts with the recognition and binding of the transcription factor TATA-binding 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, 12, 13]. The multi-subunit complex named mediator (a general transcriptional coactivator) is also necessary for the transcription
In metazoan cells, the transcription factor TBP is tightly associated to TAFs and the TBP-TAF complex is named TFIID . 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
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, 19, 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 . 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 . 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
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) ; TFIIB recognition element (BRE) ; 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 . 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
3. Characterization of ribosomal protein gene (RPG) promoters of
Schizosaccharomyces pombeand their transcription initiation mechanism
Schizosaccharomyces pombeRPG transcriptional module: the HomolD-box
The characterization of the promoter sequences of 14 RPGs from the fission yeast
|Transcription initiation and regulation of RPG expression||[36, 39, 48]|
Currently, we know that the genome of
Interestingly, HomolD-boxes in RPG promoters are broadly distributed in the
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 . 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 HomolD-box. Rrn7 showed a specific HomolD-box-binding activity and it is required for the specific transcription of RPGs containing a HomolD-box . Moreover, the GTFs and RNA pol II were required for accurate transcription initiation of a HomolD-box-containing promoter.
Rrn7 is part of the Zn-ribbon protein family related to TFIIB, including the mammalian ortholog TAF1B . 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 . Recently, it has been demonstrated that
3.3. Preinitiation complex (PIC) formation on the RPG promoters in
As stated before, RPGs that contain a HomolD-box are transcribed by the RNA pol II transcription apparatus . The formation of the PIC on a HomolD-box-containing 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 . Upon the binding of Rrn7 to the HomolD-box, the general transcription factors TBP and TFIIB are able to recognize this DNA-protein complex . 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 . 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.
4. Regulation of RPG expression in
The expression of genes containing the HomolD-box in their promoters is almost unknown. However, data from analysis of the RPG expression profiles during several biological processes in
Several promoters of
Now that the complete genome of
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
The modular architecture of the
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.
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 essential for accurate translation activity. The coordinated regulation and expression of the RPG with other ribosomal components is still poorly understood. However, in the fission yeast
In addition to the role of CK2 to modulate Rrn7 function in
6. The TCT-motif module in metazoan RPG
The analyses of insect and mammalian RPG promoters have shown the presence of a common core promoter element that is part of the poly-pyrimidine initiator (TCT)-motif family, which is a novel core promoter element necessary to initiate transcription in those genes [35, 57]. In these promoters, the transcription start site (TSS) involves the TCT motif and is positioned around −2 to +6 relative to TSS, competing with exactly the same position as the Inr. However, the features of the TCT-containing promoters are dissimilar to those Inr-containing promoters. The function of a TCT motif cannot be replaced by an Inr, and the TFIID complex cannot bind to the TCT motif . Recently, studies in
The fission yeast
This work has been funded by the Corporación Nacional de Ciencia y Tecnología de Chile (CONICYT), Fondo de Desarrollo de Ciencia y Tecnología (FONDECYT), and FONDEF (ID16I10145). We are very grateful to Mrs. Teresa Donoso for her continued support to F. U. and E. M.
Conflict of interest
The authors do not have any conflict of interest.
|CPE||core promoter element|
|DPE||downstream promoter element|
|GTF||general transcription factor|
|RNA pol||RNA polymerase|
|RPG||ribosomal protein genes|
|snoRNA||small nucleolar RNA|
|TCT||poly-pyrimidine initiator motif|
|TSS||transcription start site|