Eukaryotic Gene Regulation Transcription Factors
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- YONG YA FEN
- What are the mechanisms of bacterial transcription initiation?
The transcription initiation is dependent on the interaction between RNA polymerase and promoter. The way that RNA polymerase interacts with promoter determines the direction for transcription because it decides which DNA strand to be the template strand and the site where transcription begins (Russell, 2010). A promoter is a DNA sequence that is located at the upstream of the transcription initiation site. There are two regions within the promoter are critical for the transcription initiation. These two regions are generally referred to as -35 and -10 that are 35 and 10 base pairs upstream from the site where transcription starts which is normally numbered +1 (Lodish et al., 2000; Russell, 2010). The consensus sequences for the -35 and -10 regions are respectively 5’-TTGACA-3’ and 5’-TATAAT-3’ (Russell, 2010).
In bacteria, only one type of RNA polymerase is found for transcription of all tRNA genes, rRNA genes, and protein-coding genes (Russell, 2010). A form of RNA polymerase which is known as the holoenzyme is required for transcription initiation. The holoenzyme is made up of core enzyme which is bound to a subunit known as sigma factor (σ) (Lodish et al., 2000). The core enzyme is a form of RNA polymerase which consisting of two α (37 kDa), one β (151 kDa) and one β’ (156kDa) subunits (Lodish et al., 2000). In bacteria such as Eschericia coli, promoters can be recognized by a sigma factor, σ70 which has a molecular weight of 70,000 Da (Russell, 2010). The sigma factor ensures the stable binding of RNA polymerase only at promoter so that the transcription can be initiated at the specific or correct sites (Russell, 2010). However, sigma factor is not needed for the stages of elongation and termination in transcription (Lodish et al., 2000). The β and β’ subunits have function of polymerizing ribonucleoside triphophates (NTPs) as directed by the DNA template strand (Lodish et al., 2000). The α subunits can interact with regulatory proteins or DNA to control the frequency of transcription initiation from a specific promoter by RNA polymerase (Lodish et al., 2000).
Binding of the RNA polymerase holoenzyme to the specific promoter initiates transcription. The holoenzyme contacts the -35 region initially and then the promoter is fully bound (Russell, 2010). As the DNA is still in form of double helix, this state is known as closed promoter complex (Russell, 2010). The holoenzyme binds more tightly at -10 region of promoter and the DNA within that region are unwinded (Russell, 2010). The promoter in unwinded form is known as open promoter complex and it is necessary for polymerization to proceed (Russell, 2010). The sigma factor that contacts the promoter directly at the -35 and -10 regions enables the proper orientation of the RNA polymerase so that transcription can be started at the correct nucleotide of the gene when the RNA polymerase is bound at -10 region (Russell, 2010). At this point, the RNA polymerase has came into contact with around 75 bp of the DNA from regions of -55 to +20 (Russell, 2010). After about 10 base pairs of template have been transcribed, signal factor which is an initiation factor for transcription initiation will be released.
- How does the transcriptional control regulate gene expression in eukaryote?
Transcription initiation is primarily regulated in the gene expression of eukaryotic cells. The transcriptional process in eukaryotic cells is controlled by proteins that attach to particular regulatory sequences and modulate the RNA polymerase activity (Cooper, 2000). Expression of gene of different cell types of multicellular organisms can be regulated by the combined actions of various transcriptional regulatory proteins (Cooper, 2000). There are two important elements play role in regulating the transcription process in eukaryotes, which are cis- and trans-acting regulatory elements.
Cis-acting regulatory elements
Cis-acting regulatory elements are located at the same genetic region of expressed gene and comprised of proximal and distal types. These elements include promoter element, enhancer, and silencer.
The promoter elements are located at the upstream of the transcription initiation site. The core promoter is a region that is important for docking of the fundamental transcriptional machinery and assembly of preinitiation complex (PIC) (Maston et al., 2006). It also plays role in defining the location of the transcription initiation site and direction of transcription (Maston et al., 2006). The TATA box is the core promoter elements that is first described (Maston et al., 2006). This promoter element is located at the position between -30 and -100 regions (Berg et al., 2002). Although the TATA box is necessary in promoter region, it is not adequate for strong promoter activity (Berg et al., 2002). Promoter may contain some additional elements which are usually located between -40 and -150 regions such as CAAT box and GC box (Berg et al., 2002).
The proximal promoter is the region located at the upstream of the core promoter and it normally contains several activators binding sites (Maston et al., 2006). CpG island is one of the proximal promoter elements. CpG islands are generally 1000 base pairs (bp) long and have high guanine and cytosine base composition with little CpG depletion (Deaton & Bird, 2011). There are around 70% of annotated gene promoters found to be related with a CpG island (Deaton & Bird, 2011). CpG dinucleotides are usually unmethylated and they are associated with the housekeeping genes and many regulated genes (Maston et al., 2006). DNA methylation is related to transcriptional silencing. Methylation at CpG dinucleotides might lead to inhibition of transcription by preventing the binding of transcription factors to their recognition sequences (Maston et al., 2006). Methylated CpG dinucleotides can be specifically bound by methylation-specific binding proteins, for instance MeCP2 and histone-modifying complexes are recruited for establishing chromatin with repressive structure (Maston et al., 2006).
The enhancers are known as distal cis-acting regulatory elements which are typically distance away from transcription initiation site. Enhancers are able to regulate transcription and function independent of the orientation and distance from the promoter (Maston et al., 2006). They are composed of a cluster of transcription factor-binding sites (TFBSs) with spatial organization and orientation that work together to enhance transcription (Maston et al., 2006). Enhancer can interact with the promoter and hence stimulate transcription of target gene to the maximal level (Russell, 2010). Enhancers that are far away from the core promoter could have the DNA-looping model as the functional mechanism (Maston et al., 2006). There are some studies proposed that the preinitiation complex (PIC) formation might start at enhancer instead of the core promoter (Maston et al., 2006). Therefore, more precise control of the transcription activation timing would be allowed (Maston et al., 2006).
Enhancer and promoter elements can bind specifically with regulatory proteins which can either activate or repress transcription. If the enhancer is bound by a repressor and the promoter element is bound by an activator, the effect depends on the interaction between these regulatory proteins (Russell, 2010). If the repressor has relatively strong effect, the gene is repressed for expression and the enhancer in this case is called a silencer (Russell, 2010).
Locus control regions (LCRs) are clusters of regulatory elements that involved in regulating a gene cluster or entire locus (Maston et al., 2006). LCRs are generally composed of multiple cis-acting elements and are bound by coactivators, repressors, transcription factor, or chromatin modifiers (Maston et al., 2006). The collective activity of the components gives different effect on gene expression and LCRs have property of strong and specific enhancer activity (Maston et al., 2006).
Trans-acting regulatory elements
Trans-acting regulatory elements are encoded by gene from the other genetic regions and can bind to cis-acting elements for controlling gene expression.
RNA polymerase is an important element for producing primary transcript RNA. In eukaryotes, there are three different types of RNA polymerase, namely RNA polymerase Ι, ΙΙ, and ΙΙΙ play role in synthesizing distinct sets of RNAs. RNA polymerase Ι plays role in catalyzing the synthesis of 28S, 18S, and 5.8S rRNA molecules found in ribosomes. RNA polymerase ΙΙ catalyzes the synthesis of messenger RNAs (mRNAs) and small nuclear RNAs (snRNAs). Whereas, RNA polymerase ΙΙΙ synthesize transfer RNAs (tRNAs), 5S rRNA molecules, and snRNAs which are not produced by RNA polymerase ΙΙ.
There are three categories of factors involved in precise eukaryotic protein-coding genes transcription by RNA polymerase ΙΙ: general transcription factors (GTFs), activators, and coactivators. GTFs are needed for precise transcription initiation by assembling on the core promoter to form PIC which directs RNA polymerase ΙΙ to the site where transcription starts (Maston et al., 2006). PIC is formed by the binding of transcription factor, TFΙΙD to a complex comprised of TATA-box-binding protein (TBP) and TBP-associated factors (TAFs) (Maston et al., 2006).
Activators are the factors that stimulate initiation of transcription by recruiting PIC factors to promoter. Activators have a DNA-binding domain (DBD) and a transcription activation domain which are separated by a flexible region (Russell, 2010). Majority of the eukaryotic activators function as monomers or as dimmers – either homodimers or heterodimers (Russell, 2010). Some common structural motifs of DBDs that involved in the recognition and binding to DNA contributes to the specific-binding property of DBDs (Russell, 2010). Examples of DBD are the zinc finger, helix-turn-helix (HTH), and leucine zipper. The activation domains which do not possess readily classifiable motifs and vary greatly can stimulate initiation of transcription for up to approximately a hundredfold (Russell, 2010).
A coactivator participates in the transcription activation by interacting with activators and GTFs (Russell, 2010). Briefly, recruitment of a coactivator by activators that bound to regulatory sequences of genes leads to the recruitment of RNA polymerase ΙΙ (Russell, 2010). The RNA polymerase ΙΙ then comes into contact with the GTFs in proper orientation for starting transcription (Russell, 2010). The interactions between these trans-acting regulatory elements stimulate the initiation of transcription.
Repressor is a transcription factor that can inhibit the activation of transcription initiation. Repressor also has a DNA-binding domain and a repression domain. Binding of a repressor at a site which is near to a binding site of activator in an enhancer can establish the interaction of the repression domain of the repressor with the activation domain of the activator (Russell, 2010). Hence, the action of activator is blocked. Besides, overlapping of repression binding site and activator binding site prevents activator from binding (Russell, 2010). Recruitment of corerepressors by repressors assembles to the recruitment of coactivators by activators and this mechanism also involved in blocking activator’s action (Russell, 2010).
Insulators are approximately 0.5 to 3 kb in length and function in the manner of orientation-independent and position-dependent (Maston et al., 2006). Insulators prevent genes from being influenced by the transcriptional activity of adjacent genes. They are responsible in regulating gene expression by blocking enhancer-promoter communication and preventing the spread of repressive chromatin (Maston et al., 2006). In conclusion, the interaction between the cis- and trans- acting regulatory elements can lead to the positive and negative regulation of gene expression.
- Discuss the possible eukaryotic gene regulation that produces large number of proteins with a wide range of functional diversity.
Before performing human genome sequencing project, scientists thought that a human genomic DNA was made up by around 150,000 different genes (Kashyap & Tripathi, 2008). The estimation was based on the number of various mRNAs and other transcripts found in humans and assumed that each mRNA is transcribed from one gene (Kashyap & Tripathi, 2008). However, it was found that there were less than 25,000 genes in human after sequencing was completed (Kashyap & Tripathi, 2008). Hence, it raised important questions regarding the genomic complexity. Alternative splicing, which is a post-transcriptional control mechanism can explain this phenomenon.
A constitutive splicing usually removes introns from pre-mRNA and joins the exons together. Most of the splicing process occur within the spliceosome which is a large complex made up of five ribonucleoproteins (RNPs) (Kim et al., 2007). RNPs contain the small nuclear RNAs (snRNAs) U1, U2, U4, U5 and U6 as well as 150 proteins (Kim et al., 2007). The splicing machinery uses different signals to recognize exons and introns. The four important splice signals that recognize splice sites include the 5’ and the 3’ splice sites which are respectively at the upstream and downstream of exon-intron junctions, the branch site (BS), and the polypyrimidine tract which is at upstream of the 3’splice site (Kim et al., 2007).
Alternative splicing is a process which allows the production of a variety of mRNA molecules and proteins from a single gene (Kashyap & Tripathi, 2008; McManus & Graveley, 2011). In alternative splicing process, exon can sometimes be included or excluded from the final transcript or end of an exon can have two splice sites that can be recognized by the spliceosome (Kashyap & Tripathi, 2008). Protein isoforms with different peptide sequences as well as diverse chemical and biological activities can be produced through the alternative splicing patterns which decide the inclusion of portion of coding sequence within the mRNA (Black, 2003). Alternative splicing has been discovered to occur in animals, plants, and fungi; it is important in organism development and cellular differentiation (McManus & Graveley, 2011).
There are four types of alternative splicing of pre-mRNA. The first two types is alternative 5’ splice site and 3’ splice site selection. These types of alternative splicing result in the recognition of two or more splice sites at one end of an exon (Kim et al., 2007). The third type of alternative splicing is intron retention, in which intron can be remained in the mature mRNA molecule (Kim et al., 2007). Intron retention might be due to failure of splicing machinery to identify weak splice sites that flank short introns (Kim et al., 2007). The fourth type is exon exclusion and skipping, in which exon can be removed from the transcript together with introns (Kim et al., 2007). A single pre-mRNA that consists of exons and introns could also be spliced in more complex RNA splicing process that involved all four modes (Zheng, 2004).
Since alternative splicing contributes to the transcript-level modifications and protein-level alterations (Kashyap & Tripathi, 2008), it is strongly believed to play role in gene regulation for production large number of proteins with a wide range of functional diversity.
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