BCOR 101WEEK 12
BCOR 101WEEK 12 BCOR 101
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This 66 page Class Notes was uploaded by Katarina Fielding on Sunday April 24, 2016. The Class Notes belongs to BCOR 101 at University of Vermont taught by Amanda Yonan in Spring 2016. Since its upload, it has received 11 views. For similar materials see Genetics in BioInformatics at University of Vermont.
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Date Created: 04/24/16
Regulation of Gene Expression: Prokaryotes Chapter Fourteen Gene Expression • When a gene is transcribed and translated into functional protein it is expressed • Gene that is not being transcribed, or if it’s mRNA is not being translated, is silent • Not all genes are expressed at all times • In fact there are multiple layers of control of gene expression – tightly regulated • Responsible for development and cell differentiation into different tissues Regulation of Expression • Needs to be tightly controlled for proper function and interaction • Regulation can be mutated, producing: – Too much protein – Too little protein – Protein at wrong time or in wrong location • Phenotypes: – Disease, cancer, cell death External Conditions • Microorganisms change gene expression in response to their environment • Genes can be: – Inducible: turned on in presence of substrate – Repressible: turned off by substrate – Constitutive: always expressed, regardless • Gene expression can be controlled: – Positively: expressed only when stimulated – Negatively: expressed unless shut off Lac Operon • Adjacent regulatory genes encode multiple enzymes used in lactose metabolism • Cell doesn’t need to express these genes unless lactose is present to digest • Presence of lactose is inducer – Gene expression is inducible Lactose Breakdown Lactose is a disaccharide: Breaks down into glucose (preferred food) and galactose (which is converted into glucose) Lac Operon Three enzymes: 1. β-Galactosidase: digests lactose into monosaccharides galatose and glucose 2. Permease: allows lactose to enter cell 3. Transacetylase: removes toxic by- products of lactose digestion • All three genes are contiguous and transcribed as polycistronic mRNA Repressors • Studying mutants in order to determine how the inducer leads to gene expression, discovered only constitutive mutants – Constitutive = always expressed • Therefore, system must normally be repressed and mutants lost repression • Identified two repressor genes: 1. lacI = the repressor 2. lacO = the operator Lac Operon (wt) Regulatory Mutants Other Regulatory Mutants S - C I , I and O are just some of the possible regulatory mutations Regulatory Mutants • Repressor is a trans-acting regulator • Operator is a cis-acting regulator • Repressor is a constitutively expressed gene in bacteria • Inducer (lactose in environment) is actually an inhibitor of an inhibitor • What is advantage to this type of control? Summary of Regulatory Mutants O = mutated operator cannot be bound by repressor I = mutated repressor cannot bind operator I = “super-repressor”, mutant repressor cannot be bound by inducer (lactose) • Be able to determine whether genes (Z, Y, A) will be expressed Catabolite Repression • Catabolite is the product of metabolism • If there is enough/too much product, then there’s no reason to make more • Glucose is the main product of digesting lactose – if glucose is present, no reason to digest lactose turn Lac operon off • Glucose inhibits cAMP • cAMP naturally increases affinity for RNA Pol to Lac’s promoter Catabolite Repression Glucose cAMP cAMP RNA Pol RNA Pol transcription • What is the effect on Lactose’s promoter? • How does Glucose repress expression of Lac Operon? Catabolite Repression Trp Operon • Tryptophan is an important amino acid • Yet, if there is enough Tryptophan in organism’s diet then no reason to make it • Tryptophan represses Trp operon similar to how glucose repressed Lac operon Difference is: • Glucose inhibited a promoting factor • Tryptophan activates a repressor Trp Operon Trp Regulatory Mutations Think about: • Wild type Trp Operon with/without Trp What sort of regulatory mutations could occur and what would be their effect: • What happens when the repressor’s binding site for the Operator is mutated? • Repressor’s binding site for Trp mutated? • Mutated Operator? Attenuation • Upstream of the enzymes that synthesize Tryptophan is a leader sequence • Within the leader is another regulatory sequence known as the attenuator • Attenuator’s encode multiple codons specifying the amino acid being regulated – In this case: Tryptophan – UGG codon Coordinated Expression • Important to know that transcription and translation are occurring simultaneously in prokaryotes • While the mRNA is being made by RNA polymerase, another part of the mRNA is being translated by ribosome • When ribosome comes upon a string of UGG codons, it will be looking for Trp- charged tRNAs Attenuation • When adequate Tryptophan is present in diet/media - charged tRNA’s that match the UGG codon are present • Translation continue through the attenuator sequence • Producing a terminator hairpin which blocks transcription of Trp operon Tryptophan terminates transcription of the Trp Operon Terminator Hairpin Terminator hairpin and string of U’s Slows RNA polymerase then dissociates RNA Pol from DNA Attenuation • With no, or very small amounts of, Tryptophan in diet/media charged tRNA’s that match the UGG codon are lacking • Translation will not be able to continue through the attenuator sequence • Terminator hairpin is not formed • Transcription continues through operon Lack of Tryptophan allows transcription of the Trp operon Anti-termination Hairpin RNA polymerase doesn’t dissociate from DNA, Operon expressed Trp Operon Attenuation Lamda Phage • λ is a virus that infects bacteria cells – Bacteriophage • λ chooses between lysogeny and lysis – Temperate phage • Genetic regulation controls expression of pro-lysogenic genes vs. pro-lytic genes • Once again, gene expression is regulated by repressors, influenced by environment Should I stay or should I go? The Clash: • “If I go there will be trouble…” • “And if I stay it will be double!” cro v. cI gene expression Regulation of Gene expression Questions? Discussion Regulation of Gene Expression: Eukaryotes Chapter Fifteen Gene Expression • When a gene is transcribed and translated into functional protein it is expressed • Gene that is not being transcribed, or if it’s mRNA is not being translated, is silent • Not all genes are expressed at all times • In fact there are multiple layers of control of gene expression – tightly regulated • Responsible for development and cell differentiation into different tissues Regulation of Expression • Needs to be tightly controlled for proper function and interaction • Regulation can be mutated, producing: – Too much protein – Too little protein – Protein at wrong time or in wrong location • Phenotypes: – Disease, cancer, cell death Eukaryotes • How must gene expression be different in eukaryotic organisms? – Transcription and translation are separated – mRNA is processed before translation – Multicelluar organisms have different cell types – expression leads to differentiation – Cells interact and signal diverse expression changes continually through time • Overall control is much more complex Eukaryote = Nucleus • Eukaryotic DNA is located within nucleus • Separated from ribosomes that perform translation and produce all proteins • Therefore the proteins directly regulating transcription must be translocated into the nucleus before they can express genes • Same for protein factors that enhance or silence transcription Eukaryotes • Differential gene expression in different cell and tissue types • Also respond to cellular environment ex – White blood cells: express immunoglobins (IGG) to produce variety of antibodies – Pancreatic cells: don’t express IGG, express insulin, but only in response to blood sugar • Loss of regulation leads to developmental defects, diseases and cancer Multiple Layers of Control 1. Chromatin remodeling 2. Transcription 3. Splicing and mRNA processing 4. Transport to cytoplasm 5. Stability of mRNA 6. Translation 7. Post-translational Chromatin Remodeling • Eukaryotes have much more DNA than prokaryotes and DNA is wound around histones into chromatin • When chromatin is tightly packed RNA Pol and other proteins cannot bind DNA – Therefore genes are silenced • Chromatin must be “remodeled” to loosen histones so that other proteins can bind – Genes can then be expressed Chromatin Remodeling Proteins All of these loosen DNA =O Acetylation CH 3- • Addition of an acetyl group to histones lessen histone’s grip on DNA • Histone acetyltransferases (HAT) – Enzymes that add or remove acetyls? – Silence or express gene? • Histone deacetylases (HDAC) – Enzymes that add or remove acetyls? – Silence or express gene? CH -3 Methylation • DNA itself can be directly modified by the addition of a Methyl group • Methylation silences gene expression – DNA Methyltransferases Through two different mechanisms: 1. Inhibit the binding of transcription factors 2. Recruit chromatin remodeling proteins that tighten the chromatin of this region Epigenetics • Entire regions of chromosomes can be silenced through acetylation/methylation changes – Affecting multiple genes at once • Induces chromatin remodeling • Heterochromatin = epigenetically silenced because it is so tightly wound – Includes: telomeres, centromeres, and others cis-acting vs. trans Regulation • cis-acting are specific DNA sequences located in and around the genes they are regulating – Promoters – immediately upstream of gene – Enhancers or silencers – further away • trans-acting factors are proteins that encoded by separate genes and then bind to the DNA sequences (on any chromos) – Usually called Transcription Factors Promoters • DNA sequences that serve as: – Recognition site for RNA Pol binding – Necessary for basal levels of transcription • Located immediately upstream (5’) of gene • Contain the transcription start site • And a number of consensus sequences: – TATA box – almost always – CCAAT box, GC repeats, others – often Enhancers Differs from promoters, although these sequences also promote gene expression • Enhancers (and silencers) can be located on either side of gene, away from gene or even in an intron inside gene • Increase transcription beyond basal levels • Time and tissue specific gene expression Silencers • Short cis-acting DNA sequences • Can be found near or far from gene – Any location around or within gene • Act in a tissue or temporal way to regulate gene expression • Only difference from an enhancer sequence is that silencers repress level of transcription off a specific promoter Transcription Factors • cis-acting regulatory sites influence transcription by acting as a binding site for transcription machinery • RNA Pol binds to promoter sequences • Transcription factors are proteins that bind to enhancers and silencers, and either recruit RNA Pol or inhibit it’s binding – Therefore regulating amount of expression Transcription Factors • Transcription factors themselves are regulated so they are expressed based on: – Development time points – In specific tissue types – In response to environmental stimulation • Diverse and complex effects on transcrip: – Enhance or repress transcription – Compete for binding – based on activation, concentration or timing Transcription Factor Domains Transcription factors have two functional domains: 1. DNA binding domain • Area of protein that binds directly to DNA • Must be able to bind to the cis-acting elements upstream of transcription start 2. Activation or repression domain • Area that interacts with RNA Pol, or other proteins, to induce or repress transcription DNA Binding Domains DNA binding domains have certain amino acids and specific shapes to bind DNA: • Helix-turn-helix – Two alpha-helixes separated by specific aa’s • Zinc finger – Contains Cysteines and Histidines that bind Zn+ (cofactor) and can then bind negative DNA • Basic leucine zipper – Leucine’s can “zip” together into a dimer that is basic – interacts with acids, such as DNA Pre-initiation Complex • Some transcription factors assemble at the promoter forming a pre-initiation complex • Platform for RNA Pol to bind DNA of the promoter sequence – Usually at the TATA box Repressors • Specific name for Transcription Factors that inhibit transcription • Perform same function as in prokaryotes • But through different mechanisms: – Blocking an enhancer site – Block TFs from binding – Block the promoter and/or RNA Polymerase • Pre-initiation complex/platform doesn’t form Cell Differentiation Activators and repressors for one specific gene differentially expressed along a developmental axis Differentiation mRNA Processing • In prokaryotes the mRNA is translated immediately and without changes • In eukaryotes mRNA is highly processed before translation can occur • Processing can include: – Caps and tails – Splicing out of intronic sequences – Transporting mRNA out of nucleus to ribosomes in cytoplasm mRNA Stability • All mRNA’s are degraded eventually – Limiting the amount of translation • More stable mRNA = more translation • Therefore, stability of mRNA increases gene expression, leading to more protein • Some methods for controlling stability: – Length of poly-A tail – Inhibiting translation until correct time point – Decreasing RNase’s Alternative Splicing • Using or skipping different combinations of exons will build different proteins from same mRNA sequence – One gene can produce more than one protein RNAi • Small RNA molecules are produced by cell • Bind to single stranded mRNA – Through complementary base pairing • Lead to mRNA being degraded • Therefore, these small interfering RNAs decrease gene expression by inhibiting translation • Known as RNAi – RNA interference Induced RNAi • This process has been hijacked by scientists to remove a single protein – Or decrease it’s expression level – Although RNAi is so effective that most of the time no protein is made • Allows testing of protein’s function – By analyzing mutant phenotypes • Can also be developed as a drug: – Remove abnormal proteins – Lower expression of any overexpressed protein Controlling Translation • RNAi is one example of regulating amount of translation • As with the Lac and Trp operon, the amount of product can also control the level of expression • High concentrations of a protein shut down translation through a feedback loop • Regulatory proteins can bind to mRNA – Rather than DNA – like TFs Post-translational Control • Gene expression can even be controlled after mRNA has been translated into protein • Done by regulating: – Protein modifications activate or deactivate protein – Protein’s location within the cell – Negative feedback loop – Binding to other proteins only act as dimer or only once released Protein Folding • Proteins are not able to perform any function immediately after translation • Because they are linear polymers of aa’s • Need to fold into the correct conformation – Sequence Structure Function • Proteins that are inhibited from folding, or from folding correctly, are inactive – Therefore, gene is not truly expressed Protein Degradation • Just as with mRNA, proteins can have different levels of stability • Increase stability = longer protein functions • If protein is degraded, function is decreased or lost • Some regulatory proteins add a Ubiquitin tag to proteins so they will be degraded by the Proteasome – Effectively, decreasing gene expression Questions? Discussion
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