Kinkajou : Tell us about Regulation in Eukaryotes.
Erasmus : Gene activation is very common in eukaryotes. In multicellular organisms, different DNA must be turned off in different cells so that they can assume specialised functions. However, every cell maintains a complete copy of the DNA for the organism. This means that part of the DNA of eukaryotic cells needs to be turned on and turned off at different times in the cell’s growth or life-cycle.
Erasmus : A eukaryotic cell typically contain between 5000 and 60,000 genes. The size of the DNA does not relate to complexity.
- Human DNA contains approximately 20 000 to 25,000 genes.
- Trichomonas vaginalis a common low-grade sexually transmitted disease organism contains approximately 60,000 protein encoding genes.
- A fruit fly typically has approximately 14,000 protein coding genes.
It gets more interesting if you look at the total size of the DNA genome of the organism.
- The marbled lungfish: 150 billion base pairs
(50 times larger than that of a human being.) Australian Lungfish
- Humans: 3 billion base pairs
- Trichomonas vaginalis: 160 billion base pairs
- Fruit fly: 170 million base pairs
Complexity arises due to what the genes do and how their function is regulated.
- Some genes function all the time e.g. metabolic energy production genes
- Some genes are expressed when a cell begins differentiation and others when the cell functions in a differentiated state. For example liver cells express lipid (LDL) receptors.
- Some genes function in response to hormonal stimuli.
- Some genes function response to environmental stimuli.
Kinkajou : How is gene expression regulated?
Erasmus : There are several methods used by eukaryotes.
- The most common type of genetic regulation
- Turning on and off of mRNA formation
- Regulation of the processing of a pre-mRNA into a mature mRNA
- Regulation of the rate of Initiation
- Regulation of the modification of an immature or inactive protein to form an active protein
mRNA Processing Function
Erasmus : Transcriptional Control
Transcription begins when an RNA polymerase complex (made up of an RNA polymerase enzyme and a number of special proteins), attaches to DNA.
Eukaryotes have three RNA polymerases, known as Pol I, Pol II, and Pol III. Each polymerase has specific targets and activities, and is regulated by independent mechanisms.
Mechanisms for control of polymerase activity include:
- Control over polymerase access to the gene: this includes the functions of protein complexes such as enhancers, repressors and insulators.
- Control of Elongation of the RNA transcript. Once polymerase is bound to a promoter, it requires another set of factors to allow it to escape the promoter complex and begin successfully transcribing RNA.
- Termination of the action of the RNA polymerase.
Control over polymerase access to the gene
- One initiation system is represented by the TATA box system. Approximately 10% to 30 % of human genes contain a TATA box with the core promoting segment. The TATA box consists of a basic DNA sequence 5'-TATAAA-3' followed by generally three or more adenine bases (AAA). During transcription, the TATA binding protein (and/or other proteins linked to the TATA binding protein, (TBP)) binds to the TATA box, initiating DNA unwinding, allowing the RNA polymerase to bind nearby, and initiating transcription.
This confirmation generates a low level of transcription from what is known as the basal transcription complex.
- Other constructs can accelerate transcription. One such construct is a pattern of nucleotides with a GGCCAATCT sequence known as the CAAT box this is generally located about 70 base pairs upstream of the promoter sequence. Specific proteins called CAAT box binding proteins are required to activate this transcription accelerating complex.
- Another solution to initiate transcription is for genes to use either an initiator element or downstream promoting element (DPE). Initiator sequences have a core sequence of YYANWYY and their presence is mutually exclusive to the presence of a TATA box.
Downstream promoting element was first identified in the fruit fly genome. The general sequence is thought to be RGWYV (T). It is generally located approximately 30 take three nucleotides downstream of the transcription start site. Many genetic promoters can contain both a TATA box and a DPE.
DNA Transcription Diagram
- Another initiation system is represented by hormones which bind to DNA segments at sites called hormone “response elements”. These hormone response elements represent promoter sites in DNA. Hormones attaching the sites exert effects by initiating transcription of DNA.
- Proteins are also capable of binding to DNA initiating transcription. Typical scenarios include transcription factor proteins. One part of the protein complex is responsible for DNA binding. Another is responsible for formation of an activated complex or dimer.
Another part of the protein complex is responsible for transcriptional activation. The use of multiple different proteins with activation complex can give a variable level of transcription activation.
Kinkajou : Just how do proteins bind to DNA?
Erasmus : Several common structures are found:
- Helix-turn-helix (homeodomain) - three different planes of the helix are established and bind to the grooves of the DNA
- Zinc fingers - cysteine and Histidine residues bind to a Zn2+ ion, looping the amino acid into a finger-like chain that will rest in the grooves of DNA
- Leucine zipper - dimers result from Leucine residues at every other turn of the a-helix. When the a-helical regions form a Leucine zipper, the regions beyond the zipper form a Y-shaped region that grips the DNA in a scissors-like configuration
- Silencers and
- Enhancers or ("Enhancer-binding protein"/ complexes)
Enhancers can be located upstream downstream or even within the gene this transcription they control. Their location can be many thousands of base pairs distant from the site of their action.
One proposal for explaining their mode of action is that they may draw the DNA into a loop, facilitating recurrent transcription.
Enhancers can work even if their normal 5' to 3' orientation is flipped
Enhancers can work even if they are moved to a new location
DNA Transcription Enhancers
Kinkajou : Tell us about enhancers of transcription
Erasmus : Enhancers
- Studies of the human genome predict that an active promoter interacts with 4 to 5 enhancers. Similarly, enhancers can regulate more than one gene without linkage restriction and are said to “skip” neighbouring genes to regulate more distant ones.
- Even though infrequent, transcriptional regulation can involve elements located in a chromosome different to the one where the promoter resides. More interestingly, proximal enhancers or promoters of neighbouring genes can serve as platforms to recruit more distal elements.
DNA Trancription Enhancers
Kinkajou : Tell us about silencers of transcription
Erasmus : Silencers or ("Silencers-binding protein"/ complexes)
- Silencers have similar characteristics to enhancers but have an opposite effect. I.e. they reduce rather than accelerate transcription.
- Regulatory sequences with similar characteristics, but the opposite effect, exist. These are called silencers.
Kinkajou : Tell us about insulators of transcription
Erasmus : Insulators
- Insulators act to prevent enhancers from inappropriately binding to activating the promoters of other genes in the same region of the chromosome. The structure can be as small as 42 base pairs. They usually located between the enhancer and the promoter or between the silencer and the promoter.
- Thus their function is to prevent a gene from being influenced by the activation (or repression) of its neighbours.
- All insulators discovered so far in vertebrates work only when bound by a protein designated CTCF ("CCCTC binding factor"; named for a nucleotide sequence found in all insulators). CTCF has 11 zinc fingers.
DNA Transcription Insulators
An example of an insulator occurs in the gamma delta T-cells. The delta chain receptor gene is located close to the promoter for the alpha chain receptor gene, on chromosome 14 in humans. A “T cell” must choose between the activation or one or other of the T-cell receptors. The insulator between the alpha gene promoter and the delta gene promoter ensures that activation of one gene does not cause activation of the other gene.
Another example in mice and humans occurs within insulator between the Igf2 promoter and enhancer. The father’s allele is methylated while the mother’s allele is not. . CTCF can no longer bind to the insulator, and so the enhancer is now free to turn on the father's Igf2 promoter. (This phenomenon is called imprinting).
Methylation Forms a very important non-genetic method of activating or inactivating chromosomal genetic DNA. Defects and methylation can create diseases such as “Silver Russell Syndrome”, in humans.
In prokaryotes, the base setting of DNA transcription is “always on” or non-restrictive in the absence of modifying factors.
In eukaryotes, the base setting at DNA transcription is “always off” or restrictive. It is essential to recruit a number of factors to initiate DNA transcription to RNA. Factors called General Transcription factors (GTF) are required for all transcription events. They allow the RNA polymerase to bind DNA and open the DNA helix, but are non-specific for different promoter sites. Other transcription factors are essential.
Once a polymerase is successfully bound to a DNA template, it often requires the assistance of other proteins in order to leave the stable promoter complex and begin elongating the newly forming RNA strand. This process is called promoter escape, and is another step at which regulatory elements can act to accelerate or slow the transcription process.
Similarly, protein and nucleic acid factors can associate with the elongation complex and modulate the rate at which the polymerase moves along the DNA template.
Kinkajou : Isn’t the nuclear DNA of eukaryotes complicated by winding?
Erasmus : Regulation at the level of chromatin within the Nucleus
In a eukaryote, DNA is compacted by winding around protein histone octamers. Significant chromosomal regions can be silenced as they become inaccessible to polymerases or their activating protein complexes.
Histone packing can be altered by post-translational modification to the tails of the proteins. A number of enzymes are involved such as the histone acetyltransferases (HATs), histone methyltransferases (HMTs), and histone deacetylases (HDACs), among others. These enzymes can add or remove chemical groups such as:
- Methyl groups,
- Acetyl groups,
- Phosphates, and
Such modifications can increase or decrease chromatin compaction, sequester promoter elements, or increase the spacing between histones, allowing or denying the attachment of transcription factors or polymerase on open DNA. Histone modifications can be created by the cell or can be inherited in an epigenetic fashion from a parent.
During cell growth, cells routinely modularly switch on or off entire gene regulatory networks. Many genes are located not within operons but in regions called “Topological Association Domains” (TAD’s). TAD is contained many genes regulated by many enhancers. They serve as a large functional domain rather than a physical one.
Gene Engineering Viral Vectors
Erasmus : Post-Transcriptional Control
Most eukaryotic protein genes are split into segments. The segments of DNA coding for amino acids in the protein are called exons. The stretches of DNA intercalated between exons are known as introns. Introns can be transcribed into RNA but are not translated into proteins.
A complex of many proteins called a spliceosome is responsible for the removing of introns and the splicing of exons. It is believed that most introns begin with a GU sequence and end with an AG sequence. Exons must be spliced exactly, as even a single nucleotide error in splicing can cause a frame shift error in the reading of the RNA strand. This will result in the production of a completely different protein.
Cis splicing is the predominant method by which introns are removed from eukaryotic pre-– mRNA.
The existence of splicing in eukaryotes can create a situation where a single promoter can co-regulate genes that make separate mRNAs by alternative splicing. An mRNA may be spliced in two different ways to give rise to 2 different final mRNA products that essentially appear to be the product of two different genes, even though the final mRNA products she won a more exons. These final mRNAs are called alternative splicing products.
This may be considered an operon in the sense that two genes are expressed from the same promoter. However, these genes are not co-expressed since the production of one final mRNA excludes the production of the other.
DNA Splicing Operation
Erasmus : RNA Processing: pre-mRNA > mRNA
The steps involved in exon splicing processing:
- Synthesis of a cap segment, usually of guanine (G) attached to the 5' end of the pre-mRNA as it emerges from RNA polymerase II (RNAP II). This is essential to protect the RNA from enzyme degradation.
- Introns are removed sequentially from the pre-mRNA, and exons are spliced. It takes place as the pre-mRNA continues to emerge from RNAP II.
- A poly (A) tail (a stretch of adenine nucleotides ) is synthesised and attached to the exposed 3' end of the mRNA strand, at a site which may be remote from the end of the strand..
This completes the mRNA molecule, which is now ready for export to the cytosol.
(The remainder of the original transcript is catabolised /destroyed and the RNA polymerase detaches from the DNA.)
Other systems of post-transcriptional control include:
The generation of anti-mRNA that can be directed to inhibit a single type of mRNA molecule. Specific transcripts can hence be translationally inactivated.
Polycistronic transcription has been discovered in eukaryotes such as trypanosomes, nematodes, and flatworms. These organisms like introns so trans-splicing is the only process the pre-mRNAs undergo.
Trans-splicing involves the transfer of very a small leader, (known as the spliced leader or SL) from a short RNA donor segment (called SL RNA) to the ends of some or all of the pre-mRNAs produced. Trans-splicing in these organisms makes operons possible as it allows pre-mRNA from downstream genes to escape exo-nucleolytic degradation. Degradation of remnant mRNA is very fast and rapid, to the extent that it has been difficult to demonstrate that polycistronic mRNAs are the progenitors of mature final mRNAs.
Spliceosomes catalyse both trans-splicing and Cis-splicing.
Erasmus : Translational Control
- These regions typically at the beginning of the mRNA strand can bind proteins and other molecules to enhance or restrict ribosome binding, and hence translation. They are also capable of changing the rate of degradation of the RNA strand. Rapidly degraded strands of course are able to produce much translated protein.
Erasmus : Post-Translational Control
- Many proteins produced are actually pro-proteins. They must undergo further modification before assembly into the final product. The insulin molecule is a typical example, where alpha and beta chains are linked with sulph-hydride bridges and a protein segment must be excised to create the final functional molecule.
Erasmus : Other chemical modification systems include:
- Addition of methyl groups
- Addition of phosphoric groups
- Addition of glycosyl groups
- Instructions for packaging within the endoplasmic reticulum, prior to secretion.
Post Translation RNA Modification
Erasmus : Regulation through Transcription Factors and Enhancers
Transcription factors can undergo modification within the cytoplasm of the cell, and then be translated to the nucleus to interact with their corresponding enhancers.
Some post-translational chemical modifications include phosphorylation, acetylation, SUMOylation and ubiquitylation. Sumo are small proteins that covalently attach and detach to other proteins of modifying their functions.
There are important in many cellular processes such as: transport processes between the cytoplasm of the nucleus, transcription regulation, cell apotisers, and cell growth cycles. Ubiquitin is often used to tag proteins for degradation. Similar acts when the last four amino acids in the C-terminal and are cleaved allowing a formation of a peptide bond between the C-terminal glycine of the sumo protein and an acceptor lysine on the target protein.