Comparison of prokaryotic and eukaryotic transcription¶
Prokaryotic transcription overview
Prokaryotes have small control areas, while eukaryotes have larger control areas.
Prokaryotes have a simple three-part promoter (TTGACA...TATAAT...YRY in E. coli), while eukaryotes use many different promoter elements.
Prokaryotic RNA polymerase binds directly to the promoter region, whereas eukaryotic RNA polymerase II binds to the Pre-initiation Complex and only partially interacts with the promoters.
Eukaryotes almost always use one promoter to control transcription of one gene, while prokaryotes often use one promoter to control multiple genes in an operon. For the most part, there is no polycistronic mRNA in eukaryotes.
Translation and transcription can occur simultaneously in prokaryotes. RNA is transcribed first in eukaryotic cells, and then translated in the cytoplasm.
The direct product of transcription in a prokaryote is mRNA. There are introns in eukaryotic pre-mRNAs that are removed during mRNA processing.
In prokaryotes, sigma factor has to come off RNA polymerase so that RNA polymerase can be released from the promoter and continue transcription past promoter regions.
In eukaryotes, TFIIH (TFIIH is a kinase, which means it is a protein that puts phosphates on things) phosphorylates the CTD of RNA polymerase II, releasing it from the preinitiation complex. The RNA pol II then comes off the promoter and can start transcription.
In both prokaryotes and eukaryotes, cis-acting sites are sequences in the DNA that help RNA polymerase bind; they typically only affect transcription of genes nearby in the same DNA molecule.
Main points for eukaryotic gene regulation¶
Eukaryotic transcription overview
Eukaryotic gene regulation is mainly transcriptional and can control the expression of genes at various levels. Three RNA polymerases are involved. Pol I transcribes ribosomal RNA or large RNA. Pol II transcribes pre-cursors or pre-messenger to mRNA and Pol III is responsible for smaller RNAs. Pol 2 does not bind to the promoter directly. The chromatin structure in eukaryotes is very important for gene regulation because the availability of the promoters depends on it. In eukaryotes there are large and variable promoters. Unlike in prokaryotes, eukaryote promoters do not have the same consensus sequence in most of the genes. There is no single sequence that is found in every gene; examples of core promoter elements are the TATA box, the BRE, the INR, and the DPE (see below under "basal transcription factors"). The TATA box is only found in 12% of promoters in humans. Any Eukaryote has one core promoter element, which lead to binding of specific proteins which allows RNA polymerase to bind and start the process.
There are additional control sequences in the area up to about -200bp, known as promoter-proximal elements. The promoter is highly variable and contains multiple cis acting elements. Examples of promoter-proximal sequences are the Cat box, the GC box, octamers (oct-eight nucleotides long), and kBs.
While GC boxes act to promote transcription in eukaryotic genes they require the presence of at least two properly positioned GC boxes upstream of the promoter region.
The enhancer sites can be anywhere and are usually far apart from each other. They can be 50,000 bps away from the starting point of transcription in either direction. It has to be close enough to the starting point to fold as seen in the preinitiation Complex below.
Eukaryotic transcription can only occur if RNA polymerase is able to bind to the preinitiation complex. RNA polymerase, however, does not bind directly to the promoter as in prokaryotes. Eukaryotic DNA is super-coiled, wrapped up with proteins and packaged tightly in a manner that has a strong effect on whether a promoter will be accessible to activator proteins and even more importantly, RNA Polymerase II. If DNA is bound too tightly to protein complexes made of histones (nucleosomes), the basal transcription factors cannot bind. In order to weaken the bonds of DNA to histones the N-terminal tails of the histones (containing many lysine residues) can be acetylated. Therefore, the structure of the chromatin, which includes the DNA and histones, is important for gene regulation.
Gene regulation is currently being researched extensively in regards to developmental genes. An example of this is in muscular degenerative diseases such as Duchenne muscular dystrophy. It has been shown that satellite cells that develop the skeletal muscle of the fetus decrease with age. The Ezh2
gene which regulates the activity of many other genes has shown to decrease in expression with age. It is currently being studied whether the activation of Ezh2 could promote satellite cell production which could be used to help DMD patients(Juan et al. 2011).
Overview of initiation of transcription in eukaryotes
See video on formation of the preinitiation complex
This image shows the preinitiation complex. RNA polymerase II is only able to start transcription by binding to the preinitiation complex. RNA ploymerase II is bound to a group of proteins that assemble around the start point of transcription, the proteins provide a base for the binding of polymerase II. The enhancer proteins and promoter proteins, as shown in the figure, help to stabilize the preinitiation complex long enough for RNA polymerase II to bind. Without these enhancer proteins and promoter proteins, it is less likely that transcription will occur.
Enhancer Protein: An enhancer site can be quite some distance away from the promoter. If the DNA- binding domain binds to the enhancer site the pre-initiation complex can be formed by folding the DNA over, where the Activator Domain will bind to either a cofactor or directly to the TAFs. This is another way to stabilize the pre-initiation complex. One example of an enhancer protein is the glucocorticoid receptor. The gene for a specific receptor is transcribed and translated, forming a functional glucocorticoid receptor which can then bind to its respective hormone. Once the hormone binds, the resulting hormone-receptor complex is an active enhancer protein. The DNA-binding domain of the receptor binds to the DNA, while the active domain of the receptor binds either to a TAF or to a co-activator, both of which are parts of the preinitiation complex.
This preinitiation complex is constantly forming and coming apart in a dynamic equilibrium. It just has to stay stable long enough for RNA polymerase II to bind and begin transcription of the gene. This needed stability is provided by the above-mentioned activator proteins that bind to enhancers and promoter elements. The preinitiation complex itself is unlikely to be stable enough alone. The preinitiation complex is held together with H bonds, like practically everything we ever talk about in this course. The more activator proteins that are bound to the DNA and to the preinitiation complex, the more stable it is, and the more likely RNA pol II is to bind and transcribe the gene.
TFIID is one of the basal transcription factors, which include TFIIA, TFIIB, TFIID, and TFIIH. TAFs stand for TBP-Associated Factors. TBP stands for TATA-Binding Proteins. TFIID is composed of multiple proteins.
TFIIH is a helicase, which denatures DNA. It is also a kinase, which adds phosphate groups onto something. If that something is a protein, then the protein's conformation can change. TFIIH specifically phosphorylates the carboxy-terminal domain (CTD) of RNA polymerase II, causing a conformational change, and helping the RNA polymerase to separate itself from the preinitiation complex so it can finish transcription.
If only TFIID, TFIIH, and TBP are present, then the RNA polymerase II will still bind sometimes.
Basal transcription factors in eukaryotic initiation
Basal transcription factors bind to the promoter region in genes. RNA Polymerase cannot bind to the promoter without them. For example, the basal transcription factor TBP binds to the TATA box on the DNA. The location of the TATA box varies with the organism. This leads to the subsequent binding of other proteins to form the preinitiation complex, and RNA Pol II is among these. Basal transcription factors are always binding to the DNA, but in order for transcription to begin, Pol II must be released from the preinitiation complex. TFIID is a complex made up of proteins called TAFs. TAFs are TBP associated factors. The binding of TBP kinks the DNA and stresses hydrogen bonding, making it easier to separate the DNA strands. There are multiple core promoter elements, and different genes have different promoters (they are not all the same). The activation domain makes contact with a TAF, and the enhancers site has an activator protien that binds to it.
Eukaryotic core promoter
-TF stands for Transcription Factor and II refers to RNA polymerase II
TFIIH is one of several transcription factors that make up the preinitiation complex on the DNA in eukaryotes. When all the elements of the promoter-proximal region are bound, it stabilizes the preinitiation complex, allowing for transcription. The complex continues to build and is held together by hydrogen bonds. TFIIH is a kinase that phosphorylates the carboxy-terminal domain of RNA Pol II, releasing it from the preinitiation complex so that it can continue transcription. "Phosphorylation" in general is the process of adding one or more PO43-
groups to a molecule. Kinases like TFIIH are commonly used to phosphorylate proteins, changing their shape to activate or deactivate them.
Promoter-proximal elements are binding sites found close to the start of transcription, or within about a couple of hundred base pairs from the promoter. Some examples are CAT box, GC box, octamer, and kB. The CAT box gets its name from its consensus sequence (almost) spelling out CAT, while the GC box consensus is made up of entirely G and C bases. Like the core promoter elements, at least one of these promoter-proximal elements is found in any eukaryotic gene.
Promoter-proximal elements were originally identified through promoter mutations. When regions such as the TATA box were mutated, less transcription occurred. For each proximal promoter element there is a trans-acting protein that binds to it. For example, CTF binds to the CAT box, SP1 binds to the GC box. In the GC box, SP1 increases the rate of transcription in the cell. Oct-1, Oct-2 bind to t Octamer, and NFkB binds to kB. TBP kinks DNA when it binds to the DNA. This stresses the weak hydrogen bonds holding the DNA together and causes the strands to separate which initiates transcription.
Enhancer sites and the activators that bind them¶
Activator proteins will bind to enhancer sites on the DNA. This binding causes the DNA to bend over and attach to proteins in the pre-initiation complex, typically coactivators or TAFs. As a result, the complex is stabilized through hydrogen bonding.
Some transcription factors bind to regions of DNA that are thousands of base pairs away from the gene they control. Binding increases the rate of transcription of the gene. Enhancers can be located upstream, downstream, or even within the gene they control. One possibility is that enhancer-binding proteins — in addition to their DNA-binding site, have sites that bind to transcription factors "TF" assembled at the promoter of the gene. This would draw the DNA into a loop. Recent evidence shows that these loops are stabilized by cohesion,the same protein complex that holds sister chromatids together during mitosis and meiosis.
Histones are proteins that bind tightly to DNA and can prevent transcription. A histone is DNA wrapped twice around the nucleosome. They have positive lysine residues on their N-termini and are strongly attracted to DNA because of its highly negative phosphate backbone. If acetyl groups are added to the lysine residues on the histones, the charge is neutralized which makes it possible for SWI/SNF (chromatine remodeling protein) to nudge the nucleosomes over just enough so that the TATA box is exposed. With the TATA box exposed, TBF and other TF's can bind to it, allowing RNA polymerase to bind to the preinitiation complex and start transcription. If a histone deacetylase binds, the deacetylase will remove acetyl groups from lysines on the N-termini of histones. This would cause the positive charge of the lysine and the negative charge on the DNA to become strongly attracted to eachother, and the histones would bind tightly. This would prevent SWI/SNF from nudging aside the nucleosomes and exposing the TATA box. This, in turn, prevents RNA polymerase II from binding, and not allowing other transcription factors (TF) to bind, also preventing transcription.
Gene regulation by steroid hormones
There are two types of hormones. The first, polypeptide hormones, cannot go right through the cell wall because they are not lipid soluble. They act from outside of the cell by binding to transmembrane receptors on the cell surface. The binding brings two receptors with their associated bound proteins together to form a functional dimer, activating kinase. Kinase phosphorylates a protein which can then pass through the nuclear pore, bind to the response element, and stimulate transcription.
The second type of hormones, steroid hormones, are lipid soluble and can go right through the cell membrane because it is made of lipids. They commonly circulate in the bloodstream of many organisms. The two most common types of steroids are catabolic and anabolic. Catabolic steroids promote the degradation of tissues. Anabolic steroids promote the synthesis, or building up, of tissues. In both cases, the steroids accomplish this by their effects on regulation of gene transcription.
The steroid hormone shown in the picture is cortisol, which is a glucocorticoid. The GR stands for glucocorticoid receptor protein. The GR changes conformation when G, or glucocorticoid hormone, binds to it. The GRE is the glucocorticoid response element, which is an enhancer site on the DNA. It enhances transcription on the nearest promoter.
Once the steroid hormone binds to the receptor, the receptor becomes active and travels through the nuclear pore. Binding of the steroid results in a conformational change in the receptor. Often, an inhibitory protein is released from the GR by hormone-binding, thus unmasking the receptor's DNA-binding domain. Derepression of the DNA-binding site allows the receptor to bind to the enhancer site on the DNA strand. The binding of the receptor to the enhancer allows the transcription to occur, but without the receptor bound to the enhancer transcription will not occur. The use of steroids have their effects by regulating transcription in this manner. There is no need for a transport protein to bring a steroid into a cell.
GRE is a specific regulatory DNA sequence that mediates the effects of glucocorticoids on gene transcription through interaction with the activated glucocorticoid receptor. It is located upstream of a particular gene. A consensus sequence for one such GRE is the almost palindromic structure GGTACAnnnTGTTCT.
Read more: http://www.answers.com/topic/glucocorticoid-response-element#ixzz1cxdZZJYQ
Check out this video on the glucocorticoid mechanism of action http://www.youtube.com/watch?v=fqtaqOhGEKc&feature=related
The nuclear pore is a system all in itself as seen in this video: http://video.google.com/videoplay?docid=6374761646657730470#
There are many possible mutations that can allow for transcription with a low concentration of the steroid hormone. An example of such a mutation would be a mutation that codes for an altered protein in the nuclear pore.
The nuclear pore is made up of a complex of multiple proteins called nucleoporins.The nucleoporins allow the diffusion of larger molecules into the nucleus of the cell. It is thought that with a normal nuclear pore only an active receptor could enter into the nucleus; to make the receptor active, a steroid hormone must bind and change the conformation of the complex.This is not proven; however, if it is true then it is possible for an inactive receptor protein to enter the nucleus if one or multiple proteins were mutated in the nuclear pore.
Studies have shown that the nuclear pores actually function as transcription factors. The nuclear porins have the capacity to bind to certain genes, specifically developmentally regulated genes. For instance, when Nup98 abnormally fuses with particular gene regulatory proteins, leukemia can result. Further, high expression of particular nuclear porins have shown to be associated with other cancers (Salk Institute 2010).
Juan AH, Derfoul A, Feng X, Ryall JG, Dell'Orso S, Pasut A, Zare H, Simone JM, Rudnicki MA, Sartorelli V. Polycomb EZH2 controls self-renewal and safeguards the transcriptional identity of skeletal muscle stem cells. Genes Dev. 25:789-794.
Salk Institute for Biological Studies. 2010. Nuclear pore complexes harbor new class of gene regulators, offer clues to gene expression and cancer. Salk News website. http://www.salk.edu/news/pressrelease_details.php?press_id=406.
See alsoAlternative Splicing