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Lecture 19

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Biological Sciences
Mark Fitzpatrick

BIOA01H3 – Lecture 19 th st Week of Oct. 21 : Central Dogma Chapter 14 14.1a The Operon Is a Unit of Transcription Operon is a cluster of prokaryotic genes and the DNA sequence involved in their regulation Promoter is a region where RNA polymerase begins transcription  Another regulatory DNA sequence in operon is operator, a short segment that is binding sequence for regulatory protein  Some operons are controlled by a regulatory protein called repressor, which, when bond to DNA, decrease likelihood that genes will be transcribed; activator increases likelihood  Each operon is transcribed as a unit from promoter into single mRNA  mRNA contains codes for several proteins as a result  Cluster of genes transcribed into single mRNA called transcription unit 14.1b the lac Operon for Lactose Metabolism is transcribed When an Inducer Inactivates a Repressor  Metabolism for lactose as an energy source involves three genes: lacZ, lacY, and lacA  Three genes adjacent to one another on chromosome in order Z-Y-A; transcribed as a unit into a single mRNA starting with lacZ gene; promoter for the transcription unit is upstream of lacZ lacZ o Encodes enzyme β-galactosidase  catalyzes conversion of disaccharide sugar, lactose, into monosaccharide sugars, glucose and galactose o Sugars further metabolized by other enzymes, producing energy for cell by glycolysis and Krebs’s cycle lacY o Encodes permease enzyme that transports lactose actively into cell lacA o Encodes transacetylase enzyme, function of which more relevant to metabolism of compounds other than lactose  Cluster of genes and adjacent sequences that control their expression called the lac operon  lac operon controlled by regulatory protein termed Lac repressor  Lac repressor encoded by regulatory gene lacI, which is near lac operon & synthesized in active form  When lactose absent from medium, Lac repressor binds to operator thereby blocking RNA polymerase from binding to promoter  Repressor binding is kind of equilibrium; while bound to operator most of time, occasionally comes off  In moments when repressor not bound, polymerase can successfully transcribe  As a result, there is always low [ ] of lac operon gene products in cell  When lactose present is medium, lac operon turned all & all three enzymes synthesized rapidly 1  Occurs when lactose enters cell & low levels β-galactosidase molecules present convert some of it to allolactose, isomer of lactose  Allolactose an inducer for lac operon  binds to Lac repressor, altering shape so that repressor no longer bind to operator DNA  W/ repressor out of way, RNA polymerase then able to bind freely to promoter and transcribe three genes at dramatically elevated rate  B/c inducer molecule increases its expression, lac operon called inducible operon  As lactose used up, regulatory system switches lac operon off absence of lactose means no allolactose inducer molecules to inactivate repressor 14.1c Transcription of the lac Operon Is Also controlled by a Positive Regulatory System  Positive gene regulation system that makes expression of lac operon responsive to availability of glucose  Glucose can be used directly in glycolysis pathway to produce energy for cell however, lactose must first be converted into glucose by biochemical reactions that require energy  Net yield of energy from other sugars therefore less than that of glucose & cells will grow best if ensure the preferential metabolism of glucose whenever available  lac operon sensitive to availability of glucose through binding of activator protein CAP (catabolite activator protein)  CAP binding site on DNA, just upstream of lac promoter  When bound at this site, CAP bends DNA in ways that allow promoter region more accessible to RNA polymerase To understand how CAP binding is related to the availability of glucose, need to know that: 1) CAP is synthesized in an inactive form that can only bind to DNA after it is activated by binding with cyclic AMP (cAMP is a nucleotide that plays role in regulating cellular processes in both prokaryotic and eukaryotic cells) 2) cAMP levels are inversely related to uptake of glucose from growth medium; when glucose abundant, cAMP levels low (CAP mostly inactive) & vice versa  Together w/ negative control by Lac repressor & positive control by CAP/cAMP ensure cells express lac operon most strongly only when lactose present & glucose not 14.1d Transcription of the trp Operon Genes for Tryptophan Biosynthesis Is Repressed When Tryptophan Activates a Repressor  Tryptophan essential amino acid used in synthesis of proteins  Genes involved in tryptophan biosynthesis are coordinately controlled in an operon call trp operon  Five genes in this operon, trpA to trpE, encode enzymes for steps in tryptophan biosynthesis pathway  Upstream of trpE gene are operon’s promoter & operator sequences  Expression of trp operon controlled by Trp repressor, which is encoded by trpR gene (not nearby like lacI gene for lac operon)  In contrast to Lac repressor, Trp repressor synthesized in inactive form in which it cannot bind to operator  When tryptophan absent & must be made by cell, trp operon genes expressed  This is default state since Trp repressor inactive & cannot bind to operator, RNA polymerase can bind to promoter and transcribe operon 2  Resulting mRNA translated to produce five tryptophan biosynthetic enzymes that catalyze reactions for tryptophan synthesis  If tryptophan present, trp operon shuts off  Occurs b/c tryptophan entering cell binds to Trp repressor & activates it, which then binds to operator region & blocks RNA polymerase  For trp operon, presence of tryptophan represses expression of tryptophan biosynthesis genes; hence, operon example of repressible operon  Tryptophan acts as corepressor, regulatory molecule that combines w/ repressor to activate it & thus shut off operon Compare & Contrast 2 Operons: 1) lac operon, repressor synthesized in active form. When inducer (allolactose) present, binds to repressor & inactivates it. Operon then transcribed 2) trp operon, repressor synthesized in inactive form. When corepressor (tryptophan) present, binds to Trp repressor, activating it & blocks transcription 14.2 Regulation of Transcription in Eukaryotes 2 General Categories of Eukaryotic Gene Regulation: 1) Short term: involves regulatory events in which gene sets quickly turned on/ off in response to changes in environmental or physiological conditions in cell’s or organisms’ environment 2) Long term: involves regulatory events required for organism to develop & differentiate 14.2a In Eukaryotes, Regulation of Gene Expression Occurs at Several Levels  Complex compared to prokaryotic cells b/c nuclear DNA organized w/ histones into chromatin, & b/c multicellular eukaryotes produce large #s & diff. types of cells  Eukaryotic nuclear envelope separates processes of transcription & translation; prokaryotic cells, translation can occur while mRNA still being made  Gene expression in eukaryotes regulated at more levels; transcriptional regulation, posttranscriptional regulation, translational regulation, posttranslational regulation 14.2b Regulation of Transcription Initiation Involves the Effects of Proteins Binding to a Gene’s Promoter & Regulatory Sites Organization of a Eukaryotic Protein-Coding Gene  Immediately upstream of transcription unit is promoter  Promoter contains TATA box, sequence of ~25bp upstream of start point for transcription  TATA box has 7-bp consensus sequence 5’-TATAAAA-3’ 3’-ATATTTT-5’  Promoters w/o TATA boxes have other sequences playing similar roles  RNA polymerase II cannot recognize promoter sequence; proteins called transcription factors recognize & bind to TATA box then recruits polymerase 3  One RNA polymerase II-transcription factor complex forms, polymerase unwinds DNA & transcription begins  Adjacent to promoter but farther upstream, is promoter proximal region  contains regulatory sequence called promoter proximal elements  Regulatory proteins that bind to promoter proximal elements may stimulate or inhibit rate of transcription initiation  More distant from beginning of gene is enhancer  Regulatory proteins bind to regulatory sequences within enhancer also stimulate/inhibit rate of transcription Activation of Transcription  Initiating transcription requires proteins called general transcription factors (also called basal transcription factors) bind to TATA box  Factors recruit RNA polymerase II & orient enzyme to start transcription at correct place  Combination of general transcription factors w/ RNA polymerase II is transcription initiation complex  On its own, this complex brings low rate of transcription initiation, leading to few mRNA transcripts  Activators are regulatory proteins playing role in positive regulatory system controlling expression of one ore more genes  Activators binding to promoter proximal elements interact directly w/ general transcription factors at promoter to stimulate transcription  Housekeeping genes – genes that are expressed in all cell types for basic cellular functions i.e. glucose metabolism – have promoter proximal elements that are recognized by activators present in all cell types  DNA-binding & activation fnc of activators are properties of two domains in proteins  3D arrangement of amino acid chains within & between domains produces highly specialized regions called motifs  Several types, each w/ specialized fnc, found in proteins, including motifs that insert into DNA double helix  Motifs found in DNA-binding domains of regulatory proteins, i.e. activators, include helix- turn-helix, zinc finger, and leucine zipper  Activators binding to enhancer greatly increases transcription rates  Enhancers of diff. genes have diff. sets of regulatory sequences which bind particular activators  Coactivator (also called mediator), large multiprotein complex, forms bridge between activators at enhancer and proteins at promoter & promoter proximal region, causing DNA for form loop  Interactions between activators at enhancer, coactivator, proteins at promoter & RNA polymerase greatly stimulate transcription up to its maximal rate Repression of Transcription  In some genes, repressors oppose effect of activators, thereby blocking/reducing transcription  Final rate of transcription then depends on “battle” between activation signal & repression  Repressors in eukaryotes work in several ways; 4 1) Some bind to same regulatory sequence to which activators bind (often in enhancer), inhibiting binding of activators 2) Others bind to own specific site in DNA near where activator binds & interacts w/ activator so cannot interact w/ coactivator 3) Other repressors bind to specific sites in DNA & recruit corepressors, multiprotein complexes analogous to coactivators except they’re negative regulators, inhibiting transcription initiation Combinatorial Gene Regulation Review of key elements of transcription regulation for protein-coding gene:  General transcription factors bind to certain promoter sequences i.e. TATA box & recruit RNA polymerase II: results in basal level of transcription  Specific activators bind to promoter proximal elements & stimulate rate of transcription  Activators also bind to enhancers to greatly stimulate transcription of gene How are these events coordinated in regulation gene expression?  Gene has specific # & types of promoter proximal elements  Some may have one, but genes under complex regulatory control have many regulatory elements  Number & types of regulatory sequence in enhancer specific for each gene  Promoter proximal regions & enhancers important in regulating transcription  Each regulatory sequence in those 2 regions binds specific regulatory protein  Some regulatory proteins are activators & others repressors  If activators bind to both regulatory sequence in promoter proximal region & enhancer, transcription rate maximal  But if repressor binds to enhancer & activator binds to promoter proximal region, expression depends on relative effects of two regulatory proteins  Relatively small number of regulatory proteins control transcription of all protein-coding genes  By combining few regulatory proteins in particular ways, transcription of wide array of genes controlled  combinatorial gene regulation Consider example; two genes, each w/ activators already bound to promoter proximal elements  Maximal transcription of gene A requires activators 2, 4, 7, and 8 binding to regulatory sequences in enhancer  Maximal transcription of gene B requires activators 1, 5, 8, and 11 binding to enhancer  Looked at this another way: both gene A & gene B require activators 5 & 8 combined w/ other diff. activators for full activation Coordinated Regulation of Transcription of Genes with Related Functions  In prokaryotic operons, genes w/ related functions often clustered and transcribed from one promoter onto single mRNA  That mRNA translated to produce several proteins encoded by genes  No operons in eukaryotes, yet transcription of genes w/ related functions coordinately controlled 5  All genes coordinately regulated have same regulatory sequence  w/ one signal, transcription of all genes controlled simultaneously Consider control of gene expression by steroid hormones in mammals: Hormone is a molecule produced by one tissue and transported via the blood stream to a target tissue or tissues to alter physiological activity. Steroid is a type of lipid derived from cholesterol i.e. testosterone & glucocorticoid  Testosterone regulates expression of large number of genes associated w/ maintenance of primary & secondary male characteristics  Glucocorticoid (among other actions) regulates expression of genes involved in maintenance of concentration of glucose  Steroid hormones acts on specific target tissues in bond b/c only those cells in those tissues have steroid hormone receptors in cytoplasm that recognized & bind hormones  Steroid hormone moves through plasma membrane into cytoplasm & receptor binds to it  Hormone-receptor complex then enters nucleus & binds to specific regulatory sequences that are adjacent to genes whose expression controlled by hormone  Binding activates transcription of those genes & proteins encoded by genes synthesized rapidly  Single steroid hormone can regulate many diff. genes b/c all of genes have identical DNA sequence – steroid hormone response element – to which hormone-receptor complex binds  I.e. all genes controlled by glucocorticoid have glucocorticoid response element w/ them, therefore release of glucocorticoid into bloodstream coordinately activates transcription of genes w/ that response element 14.2c Methylation of DNA Can Control Gene Transcription  Binding proteins to DNA can regulate transcription, so can changes to physical form of DNA  In DNA methylation, enzymes add methyl group (CH ) to c3tosine bases  Methylated cytosines in promoter regions regulate transcription through process called silencing, in which transcription of genes controlled by promoters is greatly reduced  example of epigenetics, phenomenon where change in gene expression caused w/o changes in DNA sequence of that gene or of genome  DNA methylation underlies
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