gene expression and regulation - 1 we have been ......gene expression and regulation - 1 we have...

Gene Expression and Regulation - 1

We have been discussing the molecular structure of DNA and its function in DNAreplication and in transcription. Earlier we discussed how genes interact intransmission genetics, based on Mendelian principles. We will now address how geneexpression is regulated, primarily at the level of transcription and translation andsome of the ways in which we can use our current knowledge of molecular geneticsvia 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 cancerresearch all look at how genes are expressed and controlled. We will look at models ofgene expression in both prokaryotic cells and in eukaryotic cells.

It is important for cells to be able to control gene activity. We have geneticinformation for thousands of proteins. We do not want to synthesize enzymes thatare not needed, nor do we want to synthesize molecules in greater quantity thanneeded. The bacteria of our oral cavity for example, secrete a slime sheath whensucrose 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 dietis rich in tryptophan and they can absorb it from their intestinal environment. If thereis not tryptophan in the intestine, the bacterium will activate the metabolic pathwayneeded to synthesize its own tryptophan.

Prokaryotes, in general, control genes for rapid response to their environment. Byselectively activating or inhibiting gene activity, bacterial cells can take advantage ofchanging conditions.

For eukaryotes, gene regulation is tied to maintaining homeostasis – a consistentinternal environment in the face of ever-changing external conditions. Multicellularorganisms require different genes at different times of growth and development indifferent tissues. We have more complex controls of gene expression to ensure thatgenes function selectively in our different tissues.

In addition, many genes in multicellular organisms are activated only at one stage ofdevelopment, do their job, and function no more. The effects of these genes are notreversible. One of the current research interests is that of stem cells – the cell linesthat lead to the development of precise tissue types, such as skin, immune system orblood cells. At some point in development, stem cells are "programmed", do theirjob, and, as a part of their programming, may lead to programmed cell death. Thechapter in your text that looks at the genetics of development goes into this topic.


Regulating Gene ExpressionGene expression starts with transcription and “ends” with an enzyme catalyzing aparticular chemical reaction, or with a structural/metabolic protein. In brief, we cancontrol 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 thatthe typical gene codes for a polypeptide that is used to help the cell function in someway, or is a structural protein. A gene that codes for such proteins is a structuralgene.

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 control is exerted chemically in two general ways: affecting molecules thatinteract with DNA, RNA and/or the polypeptide chains, or controlling the synthesis ofan enzyme or the activity of an enzyme in the cell. In addition to transcription factorsand regulatory genes, both hormones and enzymes can affect gene expression andenzyme activity.

Binding to the DNA to Read the DNA MoleculeA first step in regulation is accessing the DNA molecule to get to its instructions. Tosee how this happens we need to revisit the DNA molecule structure and look at howa gene control molecule might attach. It is the ability of certain binding proteins toattach 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 formsgrooves – one deeper, the major groove, and one shallow, the minor groove. Thenucleotides within the major groove have hydrophobic methyl groups, hydrogenatoms, and hydrogen bond donor and acceptor sites that protrude out into thegroove. These groups create a chemical pattern specific for each of the four possiblenitrogen base pair combinations. This provides a mechanism for reading thenucleotide sequences by transcription factor proteins.

Transcription factors (the gene regulators) are characterized by specific bindingsites that bind to the DNA major groove within the double helix. Although morethan 30 regulatory protein structures are known, the actual binding sites on theregulatory proteins fall into a set of DNA-binding motifs (or domains).


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 ofthe 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 atright angles to each other.

• When binding to DNA, one of the helices (the recognition helix) fits into themajor groove and the second rests against the outside of the DNA to stabilizeand position the recognition helix.

• Regulatory proteins generally have pairs of helix-turn-helix motifs distanced 3.4nm (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), eachwith 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 twoarms of the "Y" fit into the major groove.

• The leucine zipper motif can provide for flexibility because the two helicalregions 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 thatthe α helix fits into the major groove.

• The homeodomain motif, a variant of the helix-turn-helix, is found indevelopmental switch box genes, called homeotic genes. Homeotic genesdetermine body-part identity and pattern development. (Chapter 17. pp. 346-348.)

Mutant Antennapedia Homeobox Gene


Gene Regulation MechanismsWe can now turn to some of the ways in which genes specifically are regulated. Ingeneral, it is easier to study bacterial gene regulation than eukaryotic gene regulationbecause the DNA is part of the cytoplasm and direct chemical contact is more readilyseen in the prokaryotic cell. Much of the research in gene regulation has beenaccomplished with Escherichia coli. Let's first look at some examples of prokaryotegene regulation.

Gene Regulation in Bacteria - The Operon of the Prokaryotic CellAn active gene (or group of genes) includes the DNA that will be transcribedalong with a promoter and an operator. This complex is known as the operon andwas first described in 1961 by Francois Jacob and Jacques Monod.

An operon also has an additional regulatory gene that activates (turns on) orrepresses (turns off) the operon.

The Operon Complex1. Promoter

Recognized by RNA polymerase as the place to start transcription2. Operator

Controls RNA polymerase's access to the promoter, and is usually located withinthe promoter or between the promoter and the transcribable gene

3. Structural (Transcribable) GeneCodes 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 ordeactivated when attached to its controller molecule. A controllermolecule 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 therepressor, and when removed, allows the gene to be activated. This isa repressible operon.


Inducible Operon Repressible OperonRepressor active – Gene of f Repressor active with controller

(Tryptophan) attached – Gene of fController (Lactose) removes Removing controller removesrepressor to activate gene repressor from operator t o

activate gene – Gene on

Some Examples of Prokaryotic Gene RegulationProduct Inhibition - The Tryptophan OperonA cellular product (the end result of gene activity) can function to inhibittranscription, and is an example of the feedback inhibition common in cellhomeostasis. This has been shown with tryptophan, an amino acid in E. coli. Highconcentration of tryptophan stops the transcription of the set of enzymes needed tomanufacture tryptophan in the bacterial cell. Tryptophan does so by binding to anallosteric (non-active) site on the tryptophan repressor. This alters the shape of therepressor protein so that the operator blocks the attachment of RNA polymerase tothe promoter. The tryptophan operon is an example of gene regulation ofrepressible enzymes, because the presence of the product of the metabolicpathway represses (or stops) the synthesis of the enzyme(s) needed to synthesize it.Technically, tryptophan is called a corepressor because it works with the repressorprotein to block transcription, and the tryptophan operon is a repressible operon.

Repressible Operon

Repressor off (not attached) when controller Repressor active with controlleris not on the repressor. RNA is synthesized (corepressor) on. No RNA is made


The Lactose Operon – An Inducible OperonJacob and Monod studied the operon that controls lactase synthesis in E. coli, theLactose operon. In the Lactose operon, the substrate, allolactose (an isomer oflactose), attaches to a repressor protein that normally sits on the operator region ofthe gene, inhibiting transcription by blocking RNA polymerase from attaching to thepromoter. When allolactose (the controller) binds to the repressor molecule, therepressor is removed from the gene so RNA polymerase can attach to the promoterand 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 allolactosemolecules attached to the repressor. When allolactose is no longer available to bindto the repressor protein, the repressor shuts down the promoter (by sitting on theoperator), 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 thesynthesis of the enzyme. Because allolactose induces transcription, the lactoseoperon is called an inducible operon (or in some sense, a derepressable operonbecause lactose stops the repressor from its activity).

Inducible Operon

Repressor active until controller Controller (inducer) removesremoves repressor; no RNA synthesized repressor from operator; RNA synthesized

It should be noted that both the lactose operon and the tryptophan operon exhibitblocking control of gene activity. The presence of tryptophan actively blocks theoperator. With lactose, the repressor normally blocks the operator and lactosedeactivates the negative repressor. It is common for anabolic processes to haverepressible operons and catabolic pathways to have inducible operons.

Combinations of Gene ControlsNot all genes are controlled just by repressors that function to turn genes off. Somegenes are also controlled by molecules that function to turn a gene on. Thesemolecules are called activators. Activators work to enhance the promoter'sreceptivity to RNA polymerase. Without the activator molecule present, the gene ispoorly transcribed, if at all.


The lactose inducible operon works in tandem with an activator, the cataboliteactivator protein (CAP) that monitors the amount of glucose in the cell. Sinceglucose is the preferred fuel molecule in cell respiration, high levels of glucose in thecell will stop any lac operon activity no matter how much lactose is present. There isno need to degrade lactose for fuel if there is plenty of fuel available, and the lacoperon will be inhibited.

When glucose levels are low in the cell, however, cAMP (discussed previously as asecondary messenger in cell communications) accumulates. cAMP binds to theallosteric site of CAP making CAP active.

Specifically, cAMP induces a conformational change in CAP that allows the CAP's helix-turn-helix binding motifs to bind to the DNA promoter. When active, CAP binds to asite next to the lac operon promoter and makes it easier for RNA polymerase to bindto the promoter region enhancing transcription of the lactase enzymes. (CAP is alsocalled CRP for cAMP receptor protein.)


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 lacoperon, just as cAMP is a secondary messenger for many transduction pathways.

Gene Expression and Regulation - 1 0

Eukaryotic Gene RegulationGiven some examples of how genes are regulated in bacteria, we can now turn ourattention to regulating gene activity in eukaryotic cells. Although regulating DNApolymerase'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 morecomplex controls as well. Most genes in eukaryotes are controlled by atranscription factor complex, not one or two repressor molecules. However,controls exist at virtually all levels of gene activity as mentioned earlier, not justtranscription. We can now look at some examples of how genes are controlled ateach of these levels.

• Making the DNA readable• Transcription• mRNA Processing• Translation• Protein Modification• Enzyme Activity


Control at the DNA levelDNA MethylationInactive DNA contains nucleotides (especially cytosine) that have methyl groupsattached. (The Barr body is an example of a chromosome that is highly methylated.)Most methylated DNA will remain inactive during differentiation and during therepeated cell divisions of that cell line. Methylation probably is most important inpreventing the transcription of the genes intended to be permanently turned off.Special enzymes during DNA replication methylate the "daughter" DNA strands. Theimprinting of DNA in gametes, discussed briefly during our discussion of inheritance, isan example of methylation. At least some developmental abnormalities are related toa mutation that prevents methylation. Removing methyl groups from nucleotides hasbeen shown to activate a gene.

Histone AlterationRecall that nucleosomes are formed when a set of histone proteins are wrapped inDNA when chromosomes condense. A histone covering a promoter region can blockthe 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 (-COCH3) to the histone proteinsassociated with DNA facilitates transcription. Histones with acetyl groups bind moreloosely to DNA . Transcription factors may act with enzymes that add acetyl tohistone proteins.


Gene Regulation at the Level of TranscriptionAny Eukaryotic gene has a number of regions1 . Control elements that form the Transcription Initiation Complex2 . The codable gene including introns and exons3 . 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 thetranscription factors)

• An enhancer region that stimulates the binding of RNA polymerase to thepromoter region. The enhancer region has both proximal and distal controlelements. The enhancer region is comprised of non-coding DNA that binds to thetranscription factors. A transcription factor that binds to the enhancer andactivates transcription is called an activator. Activators fold the DNA so thatthe enhancers are brought to the promoter region of the gene where they bind totranscription factors


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 combinationof control elements is what is important in gene regulation rather than the code ofa specific enhancer.

• Silencers are control elements which can inhibit transcription. A transcriptionfactor that binds to a silencer control element and blocks transcription is called arepressor.

Transcription Initiation ComplexFor transcription to occur, regulatory proteins, or transcription factors, bind to theenhancer where activator proteins have attached. Hundreds of transcription factorshave been identified in eukaryotes, and most likely are the direct control oftranscription. You will recall that the result of many signal transduction pathways isthe synthesis of a transcription factor.

The binding of activators to the enhancer folds the DNA molecule to bring activatorproteins closer to the promoter region. This attracts more transcription factors tofacilitate the job of RNA polymerase by forming a transcription initiationcomplex at the promoter.

When everything comes together, we get transcription. If a repressor has attached tothe silencer region near the enhancers, activators are prevented from binding to theenhancers, and transcription is repressed.


Hormones as Chemical Regulators of TranscriptionHormones 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 atranscription activator. The activator will be recognized by all genes that respond tothat 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 thecell membrane that activate signal transduction pathways sending transcriptionfactors into the nucleus.

Feedback Transcription RepressionJust as the product can repress transcription in prokaryotes (the tryptophanrepressor), 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, ittriggers the synthesis of an enzyme that blocks transcription of the tubulinpolypeptides. When the level of the tubulins decreases, the enzyme is deactivatedand tubulin polypeptides are produced again.


Gene Regulation at the level of mRNA ProcessingWe saw earlier that when we do mRNA processing we can get different functionalmRNA transcripts depending on which part of the primary transcript is determined tobe introns and which exons. These differences can be accomplished via alternativesplicing by the spliceosomes, thereby controlling gene activity.

For example, a common gene is used for the synthesis of calcitonin, a hormone, or thesynthesis of CGRP, a polypeptide messenger molecule. In the thyroid gland, the CGRPsection is an intron, and the gene codes for the synthesis of calcitonin. In thehypothalamus, the gene codes for CGRP, and the calcitonin information is an intron.

Two different active transport protein binding sites can be transcribed by switchingexons in a common pre-mRNA transcript. If the final mRNA contains the potassiumexon, the protein will bind and transport potassium. If a calcium ion exon is coded,the protein will bind and transport calcium.


Post Transcription Controls of Gene ActivityDuration of the mRNA TranscriptThe length of time a mRNA transcript can be read prior to degradation will affect theamount 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-Atail, followed by the degradation of the cap. Once the mRNA cap is degraded, therest of the molecule rapidly degrades.

Special A-U nucleotide sequences in the mRNA near the 3' poly-A tail typicallydetermine the degradation. Some nucleotide sequences near the 3' end are alsorecognition sites for endonucleases that promote rapid degradation of the transcript.

Translation ControlBlocking mRNA attachment to RibosomesTranslation can be controlled by regulatory proteins that block mRNA attachment tothe small subunit of the ribosome. In humans, translation of the protein ferritin thatbinds to iron for iron storage is blocked by the enzyme aconitase unless iron ispresent in the cell. Aconitase binds to a nucleotide sequence on the mRNA for ferritinpreventing attachment of the mRNA to the ribosome.

There are some regulatory proteins that are capable of blocking all translation withinthe cell. This is important in egg cell cytoplasm that is primed for protein synthesisafter fertilization, but does not want to start protein synthesis too early. Specialtranslation factors will initiate translation after fertilization. A similar translation blockoccurs in some algae that are only metabolically active when there is light.


Post Translation ProcessingOnce 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 isrecognized by huge protease enzymes called proteasomes. The tagged protein isreadily degraded within the proteasome. In cystic fibrosis, the chloride ion channelprotein 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 cellsignals and feedback responses.

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