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Chapter 14

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Biological Sciences

 Chapter 14: Control of Gene Expression  Structural and functional differences in cell types result from the presence or absence of the products resulting from expression of genes rather than the presence of absence of the genes themselves  Some products of genes, like housekeeping genes are expressed in nearly all cells, whereas the products of other genes may be found only in certain cell types at specific types under certain conditions.  14.1 Regulation of Gene Expression in Prokaryotic Cells  Prokaryotic organisms are single celled and simple with generation times measured in minutes so they undergo rapid and reversible alterations in biochemical pathways that allow them to adapt quickly to changes in the environment.  Thus transcription and translation are closely regulated in prokaryotic cells that reflect this type of life.  E. coli can be in the intestinal tract of a cow or even treated municipal water.  Lactose may be available in the water and the genes coding for enzymes needed to metabolize this energy source must be turned on. Other nutrients may be abundant in intestinal tract of cow.  Therefore genes coding for enzymes needed to manufacture the amino acid from scratch must be turned off.  A versatile and responsible control system allows the bacterium to make the most efficient use of the particular array of nutrients and energy sources available.  14.1a The Operon is a Unit of Transcription  In a typical metabolic process, several genes are involved and they must be regulated.  Example: three genes code proteins for metabolism of lactose by E.coli, but in the absence of lactose the three genes are transcribed very little whereas in the presence of lactose, the genes are transcribed quite actively. The on/off control of these genes is at the level of transcription.  Francois Jacob and Jacques Monod proposed the operon model for the control of the expression of genes for lactose metabolism in E. coli. It has been widely applicable to the regulation of gene expression in bacteria and their viruses.  An operon is a cluster of prokaryotic genes and the DNA sequences involved in their regulation.  The promoter is a region where the RNA polymerase begins transcription.  An operator is another regulatory DNA sequence in the operon; a short segment that is a binding sequence for a regulatory protein.  A gene that is separate from the operon encodes the regulatory protein.  Two types of regulatory proteins; repressor and activator  Repressor binds to the DNA and reduces the likelihood that genes will be transcribed  Activator binds to the DNA and increases the likelihood that genes will be transcribed.  However, many operons are controlled by more than one regulatory mechanism many repressors and activators control more than one operon.  Thus a complex network is formed which provides regulation of transcription, which allows responses to changing environments.  Each operon (which can have many genes) is transcribed as a unit from the promoter into a single messenger RNA (mRNA) and as a result the mRNA contains codes for several proteins.  Cluster of genes transcribed into a single mRNA is called the transcription unit.  A ribosome translates the entire mRNA from one end to the other, making each protein encoded in the mRNA  Usually, the proteins encoded by genes in the same operon, catalyze steps in the same process (similar to biochemical pathway).  14.1b The lac Operon for Lactose Metabolism is Transcribed When an Inducer Inactivates a Repressor  Jacob and Monod researched the genetic control of lactose metabolism in E. coli.  They found that metabolism of lactose an energy source involves three genes: lacZ, lacY and lacA.  They are adjacent to one another on the chromosome in the order Z-Y-A.  These genes are transcribed as a single unit unto a single mRNA starting with the Z gene; promoter for the transcription unit is upstream of lacZ.  lacZ encodes the enzyme B-galactosidase, which catalyzes the conversion of the disaccharide sugar, lactose into monosaccharide sugars glucose and galactose. They are then further metabolized for energy for glycolysis and Krebs Cycle.  lacY gene encodes permease enzyme that transports lactose actively into the cell.  lacA gene encodes a transacetylase enzyme which is relevant to metabolism of other compounds (other than lactose)  lac operon is the cluster of genes and adjacent sequences that control their expression.  The operator controls the operation of genes adjacent to it. For the lac operon, the operator is a short DNA sequence between the promoter and the lacZ gene.  Lac operon is controlled by a regulatory protein called lac repressor.  It is encoded by the regulatory gene, lacI which is nearby but separate from lac operon. It is synthesized in active form.  When lactose is absent, the Lac repressor binds to the operator, thereby blocking the RNA polymerase from binding to the promoter.  Repressor is bound to the operator most time, but it occasionally comes off and that’s when the polymerase can successfully transcribe.  As a result, there is always a low concentration of lac operon gene products in the cell.  When lactose is added to medium the lac operon is turned on and all three enzymes are synthesized rapidly because the lactose enters the cell and the low level of B-galactosidase coverts some of it to allolactose, an isomer of lactose.  Allolactose is an inducer for the lac operon so it binds to the Lac repressor, altering its shape so that the repressor can’t bind to the operator DNA.  Thus the RNA polymersase can bind freely to the promoter and transcribe the 3 genes at a large rate.  The lac operon is called an inducible operon because an inducer molecule increases its expression.  As lactose is used off, the lac operon is shut off and so the absence of lactose means that there are no allolactose inducer molecule to inactivate the repressor. (so the repressor can bind the operator and reduce transcription of the operon)  Bacterial mRNA are short lived so the quick turnover permits the cytoplasm to be cleared quickly of the mRNAs transcribed from an operon. Enzymes also have short lifetimes and degrade.  Refer to FIGURE 14.2 and 14.3 ON PAGE 310.  14.1c Transcription of the lac Operon is Also Controlled by a Positive Regulatory System  Years after Jacob and Monod proposed the negatively regulated operon, a positive gene regulation system was found. This system makes expression of the lac operon responsive to the availability of the glucose (used in cellular respiration). For this to occur, lactose must be converted to glucose.  Lac operon is sensitive to the availability of glucose through the binding of an activator protein called CAP (catoblite activator protein).  Cap binding site on DNA, upstream of lac promoter.  When CAP is bound to sit, it bends the DNA to make the promoter more accessible to RNA polymerase and transcription rate increases.  CAP is synthesized in an inactive form that can only bind to DNA after it is activated by the binding with cyclic AMP and cyclic AMP levels are inversely related to the uptake of glucose from the growth medium (when glucose is abundant, cAMP levels tend to be low and vice versa).  Refer to figure 14.4a & b  Negative control by the Lac repressor and the positive control by CAP/cAMP ensure that cells express the lac operon most strongly only when lactose is present and glucose is not.  Imagine if cells only grew on glucose. In presence of glucose, little cAMP is available to bind to CAP, and so there will be little stimulation of expression.  In absence of lactose, the Lac repressor will be bound to the operator site most of the time and very little synthesis of the lac genes will occur so expression of the lac operon will be low.  If we add lactose to the environment, the inducer, allolactose will bind to and inactivate the Lac repressor  Then RNA polymerase will bind to the promoter and transcribe the lac operon genes at a low level and expression will increase further as glucose is metabolized from surroundings.  This allows cAMP levels to rise, activated CAP to bind and the lac promoter to become even more available to RNA polymerase.  DNA binding proteins, the Lac repressor and CAP explain how negative control makes expression increase.  If the binding of a protein results in an increased gene expression, that is positive control.  Thus, the binding of the Lac repressor is a clear example of negative control; when the repression is released, the lac operon is induced and expression increases.  Whether gene expression is under negative or positive control depends on DNA- binding proteins not the available substances.  Positive gene regulation system using CAP and cAMP also regulated a large number of other operons that control the metabolism of other sugars. Glucose is always metabolized first.  A regulon is a type of regulatory system where several operons are under control of a common regulator.  14.1d Transcription of the trp Operon Genes for Tryptophan Biosynthesis Is Repressed when Tryptophan Activates a Repressor  Tryptophan is an essential amino acid used in the synthesis of proteins. If it is absent from the medium, E.coli must manufacture it. It if it is in the medium, the cell will use it.  Genes involved in tryptophan biosynthesis are coordinately controlled in an operon called the trp operon.  Five genes, trp A to trp E, code the enzymes for the steps in the biosynthesis pathway.  Upstream of the trpE gene are the operon’s promoter and operator sequences.  Trp repressor, a regulatory protein encoded by the trpR gene controls the expression of the trp operon.  In contrast, to Lac repressor, the Trp repressor is synthesized in an inactive form in which it cannot bind to the operator.  When tryptophan must be made due to its absence, the trp operon genes are expressed; this is the default state because the Trp repressor is inactive and cannot bind to the operator.  RNA polymerase can bind to the promoter and transcribe the operon and the resulting mRNA is translated to produce the five tryptophan biosynthetic enzymes that catalyze the reaction for tryptophan synthesis.  If tryptophan is present and doesn’t need to be made, the trp operon is shut off; occurs because the tryptophan entering the cell binds to the Trp repressor and activates it.  Active Trp repressor then binds to the operator of the trp operon and blocks RNA polymerase from binding to the promoter-so the operon cannot be transcribed.  Thus the trp open is a repressible operon and tryptophan acts as a corepressor, a regulatory molecule that combines with the repressor to activate it and shut off the operon.  Lac operon  Trp operon  Repressor synthesized in an  Repressor synthesized in an active form inactive form  Inducer present, it binds to  Corepressor present, it binds to repressor and inactivates it the repressor and activates it.  Inducer is allolactose  Corepressor is tryptophan  Operon is then transcribed  Active repressor blocks transcription of the open.  Inducible and repressible operons both illustrate negative gene regulation because both are regulated by a repressor that turns off gene expression when it binds DNA  14.2 Regulation of Transcription in Eukaryotes  In eukaryotes, the coordinated synthesis of proteins with related function also occurs, but without the need to organize genes under the control of a single promoter in an operon  Eukaryotic gene regulation is separated into short-term regulation and long term regulation  In short term regulation it involves events in which gene sets are quickly turn on or off in response to change in environment.  Long term regulation involves regulatory events required for an organism to develop and differentiate; only occurs in multicellular eukaryotes  14.2a In Eukaryotes Regulation of Gene Expression Occurs at Several Levels  Regulation of gene expression is more complex in eukaryotes and prokaryotes because they are complex with nuclear DNA that is organized with histones into chromatin and there is more variety.  In eukaryotes, the nuclear envelope separate the process of transcription and translation, but in prokaryotes translation can occur on an mRNA that is still being made.  Euakryotic gene expression is regulated at more levels; transcriptional regulation, translational regulation and posttranslational regulation.  14.2b Regulation of Transcription in Eukaryotes  Regulation of Gene expression is most common at transcription intiation.  Organization of a Eukaryotic Protein-Coding Gene.  See Figure 14.7 on pg 316  Promoter (upstream of transcription unit) in the diagram contains a TATA box, a sequence of 25 bp upstream of the start point for transcription plays and important role in transcription initiation in many promoters.  TATA box has the sequence  5’TATAAAA-3’  3’ATATTTT-5’  Promoters without TATA boxes have other sequences that play similar roles.  Transcription factors recognize and bind to the TATA box and then recruit the polymerase.  Once the RNA polymerase II transcription factor complex forms, the polymerase unwinds the DNA and transcription begins.  Promoter proximal region is adjacent to promoter. Further upstream and it contains regulatory sequences called promoter proximal elements.  The regulatory proteins that bind to these elements may stimulate or inhibit the rate of transcription initiation.  Enhancer is even further away and those proteins that bind to sequences within the enhancer also stimulate or inhibit the rate of transcription initiation.  HIGHLY RECOMMEND LOOK AT FIGURE 14.6 ON PAGE 316. I THINK IT’S THE ONLY THING SHE EVER TALKED ABOUT IN CHAPTER 14 OF HER LECTURES - Activation of Transcription  General Transcription Factors initiate transcription by binding to the promoter in the area of the TATA box  The transcription initiation complex which includes RNA polymerase II and generation transcription factors and allow the start transcription at the correct place.  By itself, it only brings a low rate of transcription initiation so only a few mRNA transcription are made.  Activators are regulatory proteins that play a role in the positive regulatory system that controls the expression of genes.  Activators that bind to the promoter proximal elements interact directly with general transcription factors and the promoter to stimulate transcription initiation, so many more transcripts can be synthesized.  Housekeeping genes are genes that are expressing in all cell types for basic cellular functions and they have promoter proximal elements that are recognized by activators present in all cell types.  In contrast, the genes expressed only in particular cell types have promoter proximal elements that are recognized by activators found only in those cell types.  DNA binding and activation functions of activators are properties of two distinct domains in the proteins.  Motifs are produced by 3D arrangement of amino acid chains within domain.  are found in proteins and include motifs that insert into DNA double helix  See Figure 14.9 for the three kinds  Activators binding at the enhancer increase transcription rates  A coactivator forms a bridge between the activator at the enhancer and the proteins at the promoter and promoter proximal region. > Causes DNA to form a loop.  All these activities stimulate transcription to maximum rate. - Repression of Transcription  In some genes, repressors oppose the effect of activators, thereby blocking or reducing the rate of transcription.  Final rate of transcription depends on the fight between activator and repressor signals.  Some repressors bind to the same regulatory sequence as activators (often in enhancer), so that the activator can't bind to the same site.  Other repressors have their own site to bind at on DNA; near where the activator binds and interacts with activator so it does not associate with coactivator  Lastly, some repressors bind to specific site in the DNA and recruit compressors, which are multiprotein complexes similar to coactivators except they are negative regulations, inhibiting transcription initiation.  Combinational Gene Regulation  How are elements of transcription regulation for a protein encoding gene coordinated in regulating gene expression?  Any gene has a specific number and types of promoter proximal elements.  Some genes have one regulatory element, but other genes have many regulatory elements; these values are specific for each gene  Promoter proximal regions and enhancer important in regulating the transcription of a gene.  Each regulatory sequence in each region binds to specific protein.  Some proteins are activators and some are repressors, so the overall effect on transcription depends on
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