Gene Expression and Regulation - 1 We have been discussing the molecular structure of DNA and its function in DNA replication and in transcription. Earlier we discussed how genes interact in transmission genetics, based on Mendelian principles. We will now address how gene expression is regulated, primarily at the level of transcription and translation and some of the ways in which we can use our current knowledge of molecular genetics via DNA technology to modify and alter genetic molecules. Gene expression and regulation is one of the most active areas of genetic research. Developmental biology, the biology of aging, genetic diseases research and cancer research all look at how genes are expressed and controlled. We will look at models of gene expression in both prokaryotic cells and in eukaryotic cells. It is important for cells to be able to control gene activity. We have genetic information for thousands of proteins. We do not want to synthesize enzymes that are not needed, nor do we want to synthesize molecules in greater quantity than needed. The bacteria of our oral cavity for example, secrete a slime sheath when sucrose is in their diet. When no sucrose is present, they do not secrete the slime, nor do they synthesize the enzymes that would process the sucrose. In a similar fashion, intestinal bacteria do not synthesize tryptophan if their host's diet is rich in tryptophan and they can absorb it from their intestinal environment. If there is not tryptophan in the intestine, the bacterium will activate the metabolic pathway needed to synthesize its own tryptophan. Prokaryotes, in general, control genes for rapid response to their environment. By selectively activating or inhibiting gene activity, bacterial cells can take advantage of changing conditions. For eukaryotes, gene regulation is tied to maintaining homeostasis a consistent internal environment in the face of ever-changing external conditions. Multicellular organisms require different genes at different times of growth and development in different tissues. We have more complex controls of gene expression to ensure that genes function selectively in our different tissues. In addition, many genes in multicellular organisms are activated only at one stage of development, do their job, and function no more. The effects of these genes are not reversible. One of the current research interests is that of stem cells the cell lines that lead to the development of precise tissue types, such as skin, immune system or blood cells. At some point in development, stem cells are "programmed", do their job, and, as a part of their programming, may lead to programmed cell death. The chapter in your text that looks at the genetics of development goes into this topic.
Gene Expression and Regulation - 2 Regulating Gene Expression Gene expression starts with transcription and ends with an enzyme catalyzing a particular chemical reaction, or with a structural/metabolic protein. In brief, we can control the expression of a gene at any of the levels of gene activity. Making the DNA readable Transcription mrna Processing Translation Protein Modification Enzyme Activity Before we go too far, we need to do some preparation work (naturally). Recall that the typical gene codes for a polypeptide that is used to help the cell function in some way, or is a structural protein. A gene that codes for such proteins is a structural gene. Other genes control how much of a polypeptide gets formed and when it gets formed. Such genes are regulatory genes. Some regulatory genes code for small polypeptides that control how other genes get expressed. These polypeptides are called transcription factors. Another type of regulatory gene is a piece of DNA that a transcription factor binds to. These regulatory sites of DNA do not actually code for any protein.
Gene Expression and Regulation - 3 Gene control is exerted chemically in two general ways: affecting molecules that interact with DNA, RNA and/or the polypeptide chains, or controlling the synthesis of an enzyme or the activity of an enzyme in the cell. In addition to transcription factors and regulatory genes, both hormones and enzymes can affect gene expression and enzyme activity. Binding to the DNA to Read the DNA Molecule A first step in regulation is accessing the DNA molecule to get to its instructions. To see how this happens we need to revisit the DNA molecule structure and look at how a gene control molecule might attach. It is the ability of certain binding proteins to attach to the DNA molecule at precise locations that provides for gene regulation. How do these binding proteins recognize the appropriate binding site? Recall in our discussion of the DNA double helix, that, as it coils, it automatically forms grooves one deeper, the major groove, and one shallow, the minor groove. The nucleotides within the major groove have hydrophobic methyl groups, hydrogen atoms, and hydrogen bond donor and acceptor sites that protrude out into the groove. These groups create a chemical pattern specific for each of the four possible nitrogen base pair combinations. This provides a mechanism for reading the nucleotide sequences by transcription factor proteins. Transcription factors (the gene regulators) are characterized by specific binding sites that bind to the DNA major groove within the double helix. Although more than 30 regulatory protein structures are known, the actual binding sites on the regulatory proteins fall into a set of DNA-binding motifs (or domains).
Gene Expression and Regulation - 4 There are four common binding protein domains. Helix-turn-helix Homeodomain Leucine Zipper Zinc Finger The helix-turn-helix motif is a protein helix with a bend. The helical region of the motif fits into the DNA major groove. The helix-turn-helix motif is formed from a region of the polypeptide that has two α helices with a non-helix "turn" between them. The two helices are held at right angles to each other. When binding to DNA, one of the helices (the recognition helix) fits into the major groove and the second rests against the outside of the DNA to stabilize and position the recognition helix. Regulatory proteins generally have pairs of helix-turn-helix motifs distanced 3.4 nm (one DNA coil) apart. Pairing of the motifs adds more strength. Tryptophan binding site with paired helix-turn-helix The leucine zipper motif has a coiled double a helix (two protein subunits), each with a hydrophobic, mostly leucine "zipper" at the end. The two leucine ends interact to hold the two subunits together while the rest of the two subunits are apart. This results in a "Y" shape, in which the two arms of the "Y" fit into the major groove. The leucine zipper motif can provide for flexibility because the two helical regions of the two arms of the "Y" can be very different. In the zinc motif zinc atoms link an α helix to a β sheet pleated protein so that the α helix fits into the major groove. The homeodomain motif, a variant of the helix-turn-helix, is found in developmental switch box genes, called homeotic genes. Homeotic genes determine body-part identity and pattern development. (Chapter 17. pp. 346-348.) Mutant Antennapedia Homeobox Gene
Gene Expression and Regulation - 5 Gene Regulation Mechanisms We can now turn to some of the ways in which genes specifically are regulated. In general, it is easier to study bacterial gene regulation than eukaryotic gene regulation because the DNA is part of the cytoplasm and direct chemical contact is more readily seen in the prokaryotic cell. Much of the research in gene regulation has been accomplished with Escherichia coli. Let's first look at some examples of prokaryote gene regulation. Gene Regulation in Bacteria - The Operon of the Prokaryotic Cell An active gene (or group of genes) includes the DNA that will be transcribed along with a promoter and an operator. This complex is known as the operon and was first described in 1961 by Francois Jacob and Jacques Monod. An operon also has an additional regulatory gene that activates (turns on) or represses (turns off) the operon. The Operon Complex 1. Promoter Recognized by RNA polymerase as the place to start transcription 2. Operator Controls RNA polymerase's access to the promoter, and is usually located within the promoter or between the promoter and the transcribable gene 3. Structural (Transcribable) Gene Codes for the needed protein 4. Regulatory Gene The regulatory gene usually codes for a repressor protein. The regulatory gene is located apart from the operon. Repressors work with controller molecules. A repressor can be active when attached to its controller molecule or deactivated when attached to its controller molecule. A controller molecule is typically a substance in the cell. A controller may function to deactivate the repressor that is blocking the gene by attaching to the repressor (hence stopping its repression) This is called an inducible operon. A controller may be a substance that is normally attached to the repressor, and when removed, allows the gene to be activated. This is a repressible operon.
Gene Expression and Regulation - 6 Inducible Operon Repressor active Gene off Controller (Lactose) removes repressor to activate gene Repressible Operon Repressor active with controller (Tryptophan) attached Gene off Removing controller removes repressor from operator t o activate gene Gene on Some Examples of Prokaryotic Gene Regulation Product Inhibition - The Tryptophan Operon A cellular product (the end result of gene activity) can function to inhibit transcription, and is an example of the feedback inhibition common in cell homeostasis. This has been shown with tryptophan, an amino acid in E. coli. High concentration of tryptophan stops the transcription of the set of enzymes needed to manufacture tryptophan in the bacterial cell. Tryptophan does so by binding to an allosteric (non-active) site on the tryptophan repressor. This alters the shape of the repressor protein so that the operator blocks the attachment of RNA polymerase to the promoter. The tryptophan operon is an example of gene regulation of repressible enzymes, because the presence of the product of the metabolic pathway represses (or stops) the synthesis of the enzyme(s) needed to synthesize it. Technically, tryptophan is called a corepressor because it works with the repressor protein to block transcription, and the tryptophan operon is a repressible operon. Repressible Operon Repressor off (not attached) when controller is not on the repressor. RNA is synthesized Repressor active with controller (corepressor) on. No RNA is made
Gene Expression and Regulation - 7 The Lactose Operon An Inducible Operon Jacob and Monod studied the operon that controls lactase synthesis in E. coli, the Lactose operon. In the Lactose operon, the substrate, allolactose (an isomer of lactose), attaches to a repressor protein that normally sits on the operator region of the gene, inhibiting transcription by blocking RNA polymerase from attaching to the promoter. When allolactose (the controller) binds to the repressor molecule, the repressor is removed from the gene so RNA polymerase can attach to the promoter and the genes that code for the three enzymes to digest lactose can be transcribed. When the enzymes are synthesized, the lactose is degraded, including the allolactose molecules attached to the repressor. When allolactose is no longer available to bind to the repressor protein, the repressor shuts down the promoter (by sitting on the operator), which stops transcription. The lactose operon is an example of gene regulation of inducible enzymes, because the presence of the substrate of the metabolic pathway can induce the synthesis of the enzyme. Because allolactose induces transcription, the lactose operon is called an inducible operon (or in some sense, a derepressable operon because lactose stops the repressor from its activity). Inducible Operon Repressor active until controller removes repressor; no RNA synthesized Controller (inducer) removes repressor from operator; RNA synthesized It should be noted that both the lactose operon and the tryptophan operon exhibit blocking control of gene activity. The presence of tryptophan actively blocks the operator. With lactose, the repressor normally blocks the operator and lactose deactivates the negative repressor. It is common for anabolic processes to have repressible operons and catabolic pathways to have inducible operons. Combinations of Gene Controls Not all genes are controlled just by repressors that function to turn genes off. Some genes are also controlled by molecules that function to turn a gene on. These molecules are called activators. Activators work to enhance the promoter's receptivity to RNA polymerase. Without the activator molecule present, the gene is poorly transcribed, if at all.
Gene Expression and Regulation - 8 The lactose inducible operon works in tandem with an activator, the catabolite activator protein (CAP) that monitors the amount of glucose in the cell. Since glucose is the preferred fuel molecule in cell respiration, high levels of glucose in the cell will stop any lac operon activity no matter how much lactose is present. There is no need to degrade lactose for fuel if there is plenty of fuel available, and the lac operon will be inhibited. When glucose levels are low in the cell, however, camp (discussed previously as a secondary messenger in cell communications) accumulates. camp binds to the allosteric site of CAP making CAP active. Specifically, camp induces a conformational change in CAP that allows the CAP's helixturn-helix binding motifs to bind to the DNA promoter. When active, CAP binds to a site next to the lac operon promoter and makes it easier for RNA polymerase to bind to the promoter region enhancing transcription of the lactase enzymes. (CAP is also called CRP for camp receptor protein.)
Gene Expression and Regulation - 9 Active CAP attached to Lac promoter Activation of the Lac operon relative to available fuels CAP functions in many operons dealing with catabolism in E coli, not just the lac operon, just as camp is a secondary messenger for many transduction pathways.
Gene Expression and Regulation - 1 0 Eukaryotic Gene Regulation Given some examples of how genes are regulated in bacteria, we can now turn our attention to regulating gene activity in eukaryotic cells. Although regulating DNA polymerase's ability to fit into the DNA to start transcription works for prokaryotes, the need for many genes to interact in eukaryote cells suggests a need for more complex controls as well. Most genes in eukaryotes are controlled by a transcription factor complex, not one or two repressor molecules. However, controls exist at virtually all levels of gene activity as mentioned earlier, not just transcription. We can now look at some examples of how genes are controlled at each of these levels. Making the DNA readable Transcription mrna Processing Translation Protein Modification Enzyme Activity
Gene Expression and Regulation - 1 1 Control at the DNA level DNA Methylation Inactive DNA contains nucleotides (especially cytosine) that have methyl groups attached. (The Barr body is an example of a chromosome that is highly methylated.) Most methylated DNA will remain inactive during differentiation and during the repeated cell divisions of that cell line. Methylation probably is most important in preventing the transcription of the genes intended to be permanently turned off. Special enzymes during DNA replication methylate the "daughter" DNA strands. The imprinting of DNA in gametes, discussed briefly during our discussion of inheritance, is an example of methylation. At least some developmental abnormalities are related to a mutation that prevents methylation. Removing methyl groups from nucleotides has been shown to activate a gene. Histone Alteration Recall that nucleosomes are formed when a set of histone proteins are wrapped in DNA when chromosomes condense. A histone covering a promoter region can block the assembly of transcription factor complexes needed for transcription. Nucleosomes do not block activators or RNA polymerase, however. On the other hand, adding an acetyl group (-COCH 3 ) to the histone proteins associated with DNA facilitates transcription. Histones with acetyl groups bind more loosely to DNA. Transcription factors may act with enzymes that add acetyl to histone proteins.
Gene Expression and Regulation - 1 2 Gene Regulation at the Level of Transcription Any Eukaryotic gene has a number of regions 1. Control elements that form the Transcription Initiation Complex 2. The codable gene including introns and exons 3. Termination Signals which end transcription The Control Elements of the Transcription Initiation Complex consists of: A specific promoter region within the control elements that indicates the starting point for transcription (and contains the TATA sequence recognized by one of the transcription factors) An enhancer region that stimulates the binding of RNA polymerase to the promoter region. The enhancer region has both proximal and distal control elements. The enhancer region is comprised of non-coding DNA that binds to the transcription factors. A transcription factor that binds to the enhancer and activates transcription is called an activator. Activators fold the DNA so that the enhancers are brought to the promoter region of the gene where they bind to transcription factors
Gene Expression and Regulation - 1 3 There are duplications of nucleotide sequences in the enhancer region of genes; only about a dozen small sequences are known. It is believed that the combination of control elements is what is important in gene regulation rather than the code of a specific enhancer. Silencers are control elements which can inhibit transcription. A transcription factor that binds to a silencer control element and blocks transcription is called a repressor. Transcription Initiation Complex For transcription to occur, regulatory proteins, or transcription factors, bind to the enhancer where activator proteins have attached. Hundreds of transcription factors have been identified in eukaryotes, and most likely are the direct control of transcription. You will recall that the result of many signal transduction pathways is the synthesis of a transcription factor. The binding of activators to the enhancer folds the DNA molecule to bring activator proteins closer to the promoter region. This attracts more transcription factors to facilitate the job of RNA polymerase by forming a transcription initiation complex at the promoter. When everything comes together, we get transcription. If a repressor has attached to the silencer region near the enhancers, activators are prevented from binding to the enhancers, and transcription is repressed.
Gene Expression and Regulation - 1 4 Hormones as Chemical Regulators of Transcription Hormones can function as chemical regulators of gene expression in their target cells. A steroid hormone can bind to a receptor protein in the cytosol that then acts as a transcription activator. The activator will be recognized by all genes that respond to that particular hormone. For example: Bird egg albumin synthesis is promoted by an estrogen-protein complex that binds near the enhancer region of the albumin gene. The molting gene in insect larvae is controlled by a hormone that activates a regulatory protein on the gene. The androgen receptor in human males is essential for testosterone to function. Non-steroid hormones and other signal molecules also trigger protein receptors in the cell membrane that activate signal transduction pathways sending transcription factors into the nucleus. Feedback Transcription Repression Just as the product can repress transcription in prokaryotes (the tryptophan repressor), a product can activate enzymes that block transcription in eukaryotes. For example: Microtubules are synthesized from two polypeptides, α-tubulin and β-tubulin. When a cell has adequate amounts of the polypeptides for microtubule synthesis, it triggers the synthesis of an enzyme that blocks transcription of the tubulin polypeptides. When the level of the tubulins decreases, the enzyme is deactivated and tubulin polypeptides are produced again.
Gene Expression and Regulation - 1 5 Gene Regulation at the level of mrna Processing We saw earlier that when we do mrna processing we can get different functional mrna transcripts depending on which part of the primary transcript is determined to be introns and which exons. These differences can be accomplished via alternative splicing by the spliceosomes, thereby controlling gene activity. For example, a common gene is used for the synthesis of calcitonin, a hormone, or the synthesis of CGRP, a polypeptide messenger molecule. In the thyroid gland, the CGRP section is an intron, and the gene codes for the synthesis of calcitonin. In the hypothalamus, the gene codes for CGRP, and the calcitonin information is an intron. Two different active transport protein binding sites can be transcribed by switching exons in a common pre-mrna transcript. If the final mrna contains the potassium exon, the protein will bind and transport potassium. If a calcium ion exon is coded, the protein will bind and transport calcium.
Gene Expression and Regulation - 1 6 Post Transcription Controls of Gene Activity Duration of the mrna Transcript The length of time a mrna transcript can be read prior to degradation will affect the amount of final product, which will affect cell activity. Some mrna lasts for weeks; some for hours. In general, mrna degradation starts with the breakdown of its poly-a tail, followed by the degradation of the cap. Once the mrna cap is degraded, the rest of the molecule rapidly degrades. Special A-U nucleotide sequences in the mrna near the 3' poly-a tail typically determine the degradation. Some nucleotide sequences near the 3' end are also recognition sites for endonucleases that promote rapid degradation of the transcript. Translation Control Blocking mrna attachment to Ribosomes Translation can be controlled by regulatory proteins that block mrna attachment to the small subunit of the ribosome. In humans, translation of the protein ferritin that binds to iron for iron storage is blocked by the enzyme aconitase unless iron is present in the cell. Aconitase binds to a nucleotide sequence on the mrna for ferritin preventing attachment of the mrna to the ribosome. There are some regulatory proteins that are capable of blocking all translation within the cell. This is important in egg cell cytoplasm that is primed for protein synthesis after fertilization, but does not want to start protein synthesis too early. Special translation factors will initiate translation after fertilization. A similar translation block occurs in some algae that are only metabolically active when there is light.
Gene Expression and Regulation - 1 7 Post Translation Processing Once a polypeptide is synthesized it can be altered by processing. Many metabolic proteins are non-functional until activated by other molecules. Hydrolytic enzymes, in particular, are synthesized in inactive forms. Membrane recognition proteins must have additional molecules attached to them before they function. Regulation can also take place when proteins are moved from one part of a cell to another, or when exported from the cell. Proteins targeted for degradation are tagged by a tiny protein, ubiquitin, which is recognized by huge protease enzymes called proteasomes. The tagged protein is readily degraded within the proteasome. In cystic fibrosis, the chloride ion channel protein gets tagged and degraded before reaching the plasma membrane. Proteasomes Although just a few examples of how genes can be controlled have been illustrated, we can see that gene control is a complex interaction of internal and external cell signals and feedback responses.