2. MARCO TEÓRICO
2.5 MÉTODOS PARA EVALUAR EL SISTEMA DE CONTROL INTERNO
In eukaryotes there are 3 different RNA polymerases that transcribe different classes of genes. RNA polymerase I transcribes 18S and 28S RNA (Grummt,
2003); RNA polymerase II transcribes mRNA (Smale and Kadonaga, 2003); and RNA polymerase III transcribes 5S ribosomal RNA, tRNA and other small catalytic RNAs (Schramm and Hernandez, 2002). As RNA polymerase II is the polymerase that transcribes protein coding RNA transcripts, only this polymerase will be focused on.
Transcription of a specific gene requires RNA pol II to bind to genomic DNA 5’ to the ATG translation initiation codon, at a region known as the core promoter. A core promoter is defined as ‘the minimal stretch of contiguous DNA sequence that is sufficient to direct accurate initiation of transcription by the RNA polII machinery’ (Butler and Kadonaga, 2002). Essentially, a core promoter is the minimal consensus that through interactions with transcription factors is able to facilitate transcription. Binding of RNA polII to the core promoter is facilitated by trans-acting general transcription factors (GTFs) that together make up the pre- initiation complex (PIC), and is the culmination of associations between cis- acting transcription factors that bind to proximal promoters, silencers, enhancers or insulators, and chromatin remodelling factors (Baumann et al., 2010).
1.8.1 Features of RNA Polymerase II Promoters
There are 2 types of RNA pol II promoters, ones that contain a TATA box and ones that do not (TATA-less). A TATA box (or Goldberg-Hogness box after its discoverers) is a core sequence of TATAAA that was initially identified as being present 25-30 bp upstream of the transcription start site in almost all RNA pol II transcribed genes examined from mammalian, viral and Drosophila protein coding genes (Smale and Kadonaga, 2003; Baumann et al., 2010). However, more recent analysis of 1031 genes by cap analysis of gene expression (CAGE) showed only approximately 32% of genes contained a TATA box within their promoters (Suzuki et al., 2001), with this figure decreasing to as low as 10% in a separate bioinformatic analysis study (Zhu et al., 2008). Further CAGE sequencing methods have revealed over 70% of TATA boxes are located between -33 and -28 from the actual transcriptional start site (TSS), with the
Another common feature of RNA pol II associated promoters is an initiator element (Inr) that encompasses the actual transcription start site, and is present in up to 85% of promoters (Suzuki et al., 2001). It has the general consensus
sequence Py2-A-N-A/T-Py2, (Py=pyrimidine, N=any base) with transcription
commonly initiating from the ‘A’ (Smale and Kadonaga, 2003; Baumann et al., 2010). Inrs are found in both TATA and TATA-less promoters. When the Inr is located ~30bp from the TATA box, the two elements act synergistically to enhance transcription, however, both can also act independently to initiate transcription with TATA being predominant over Inr (O'Shea-Greenfield and Smale, 1992). When independent of each other TATA initiates transcription ~25bp downstream and Inr initiates at its specific nucleotide within its consensus sequence (Smale and Kadonaga, 2003). The Inr is recognised by a complex of TAF1 and 2, which are part of the complex that includes TBP that form the general transcription factor TFIID (Chalkley and Verrijzer, 1999). In
vitro, RNA pol II is able to weakly bind to the Inr; the interaction and
transcription enhanced through the addition of TFIID, and TFIID is required even in promoters that lack a TATA consensus (Carcamo et al., 1989; Carcamo et al., 1991).
Downstream promoter elements (DPE) are also found in up to 85% of gene promoters. The DPE is found at exactly +28 to +32 relative to the initiating A
within the Inr, has the general consensus A/G(+28)-G-A/T-C/T-G/A/C(+32) and is
often only found in TATA-less promoters (Kadonaga, 2002; Butler and
Kadonaga, 2002). In Drosophila, the DPE is roughly found at the same frequency as TATA boxes, however in humans there is no correlation between the presence of a TATA sequence and a DPE (Kutach and Kadonaga, 2000; Gershenzon and Ioshikhes, 2005). TFIID, through its TAF6 and 9 subunits, binds the DPE (Burke and Kadonaga, 1997).
The TFIIB recognition element (BRE) is the only other characterised core promoter motif that is bound by a general transcription factor other than TFIID. The BRE consensus sequence is G/C-G/C-G/A-C-G-C-C and is immediately upstream of the TATA (Lagrange et al., 1998). Contradictory observations have been published regarding the function of the BRE. Lagrange et al identified TFIIB binding to the BRE enhanced formation of the transcription initiation complex (Lagrange et al., 1998). However, Evans et al observed an inhibitory effect on basal transcription which was reversed by addition of a transcriptional activator, and this observation corroborated with previous studies that identified mutations of the (then uncharacterised) BRE sequence directly upstream of the TATA box
that increased levels of transcription (Evans et al., 2001). It was more recently discovered that there are BREs downstream of the TATA box, with the consensus G/A-T-T/G/A-T/G-G/T-T/G-T/G (Deng and Roberts, 2005). As such the original
upstream BRE is referred to as BREU and the more recent downstream one as
BRED. The effect of BRED is promoter dependent; it has a positive increase on
transcription in promoters that lack a BREU but a negative effect on transcription
in promoters that also contain a BREU (Deng and Roberts, 2006). The two
different BREs are bound by different binding domains within TFIIB (Deng and Roberts, 2005).
GC boxes are present in almost all gene promoters (Suzuki et al., 2001). Studies on the DNA tumour Simian virus 40 (SV40) to identify sequences essential for its transcriptional activation identified three 21bp repeats, within which were 6 repeated GC rich repeats of GGGCGG. This gave rise to the GC box general consensus sequence of 5'-G/T-G/A-GGCG-G/T-G/A-G/A-C/T-3', which when present were found to be critical for transcriptional activation (Dynan et al., 1985; Gidoni et al., 1985; Imataka et al., 1992). Further studies revealed that the transcription factor Sp1 binds to these GC boxes and activates transcription (Briggs et al., 1986; Dynan and Tjian, 1983).
The final feature of gene promoters to be described is CpG Islands, which will subsequently be shown to be a critical feature of the LIMD1 promoter and thus gene expression. These are short DNA sequences approximately 1kb in length that contain a high frequency of 5’-CG-3’ dinucleotides, further referred to as CpG, and are associated with up to 100% of all housekeeping genes and approximately 70% of genes in total (Saxonov et al., 2006; Illingworth and Bird, 2009). Promoters of genes that contain one or more CpG Islands often do not contain TATA boxes or DPEs, however they are usually rich in GC boxes (both in 5’ and 3’ CpG Islands) that harbour multiple Sp1 binding sites (Gardiner-Garden and Frommer, 1987). CpG Islands will be discussed in more detail later on.
Figure 1.9: Schematic diagram of a RNA Pol II promoter. A typical transcribed gene
contains a 5’ promoter region that contains elements to facilitate transcription through the binding of transcriptional activators. Up to 70% of all genes contain a CpG Island within their promoter that may harbour multiple GC boxes for Sp1 binding. Some promoters may also contain a TATA box, where the TATA binding protein (TBP) binds or an upstream or downstream TFIIB recognition element (BREU/D) for binding of TFIIB. Both TBP and TFIIB recruit other general transcription factors and RNA Pol II to form the pre- initiation complex and initiate transcription from within the initiator element (Inr). Often in TATA-less promoters, the presence of a downstream promoter element (DPE) facilitates the recruitment of TFIID and the PIC.
Assembly of the general transcription factors and RNA pol II into a pre-initiation complex onto the DNA is the preceding step to transcription. The presence of TATA boxes and other promoter elements helps guide the transcriptional machinery to the promoter. However, in order for the Pol II basal machinery to bind to the promoter, the DNA needs to be in an accessible open or active conformation.
1.8.2 Active Transcription requires an Open Euchromatin Structure In its native state DNA is tightly packaged around nucleosomes, which are octomers of histone proteins that has ~146bp of DNA wrapped around. The N terminal tails of Histone2B and H3 protrude through the grooves of the DNA where lysines and argenines interact with the phosphate groups of the DNA to facilitate the packaging (Ruthenburg et al., 2007; Schnitzler, 2008). Post translational modifications to the histone tails alters the affinity of the histones for the DNA, and allows for the DNA to alter between a tightly packaged, transcription factor inaccessible heterochromatin structure, and a more open and accessible euchromatin conformation. When referring to histone modifications a standardised nomenclature is used. The first part of a reference is the histone (e.g. H3), the next letter is the amino acid being modified, and the final number is the residue of the amino acid; e.g. H3K9 refers to a modification of lysine 9 of histone H3.