Chapter 8Control of gene expression• Exam next TuesdayThis is vital to understand-some mutations that cause cancer and other diseases-ways to improve crop yields, alter levels of key proteinsin organisms used for biofuels-ways to metabolically engineer organisms for drug andnutrient production-basic aspects of development and evolutionObjectives:Understand the basic mechanisms of gene expression inprokaryotes and eukaryotesBe#able#to:• Draw and describe the basic steps involved in cloning aplant or animal• List 6 different steps where the activity of a protein can becontrolled• Describe 3 types of DNA-binding motifs in proteins• Explain the basic mechanism of the bacterial trp operon• Explain the mechanism of the bacterial lac operon• Be able to predict the expression of the lac operon underhigh or low glucose, high or low lactose, and in thepresence or absence of mutations of key regulatorycomponents, and be able to defend your answer• Compare and contrast bacterial and eukaryotic transcriptionregulation• Compare and contrast promoters and enhancers• Design a gene latch circuit – once a signal turns the geneon, the gene stays onCells from the same organism can be so…different!How does this happen?08_02_genetic.instruction.part1Cloning shows that different cell types contain the same DNACell differentiation must be due to changes in gene expressionFirst cat was cloned at A&M named “cc”Epigenetic effects may prevent efficient cloningof most mammals from somatic cells.Gene expression can be regulated at many stepsBut transcriptional control is paramountEarly stage regulation – very economical, but takes timeLate stage regulation – very fast, but not necessarily economical.Control of transcription initiationBinding of regulatory proteins to regulatory DNA sequencesacts as a switch to control transcription initiation (binding ofRNA polymerase to a promoter)Regulatory DNA sequences:May be as small as 10 base pairsMay be longer than 10,000 base pairs (eukaryotes)Usually located upstream of initiation siteGene regulatory proteins:Bind to regulatory DNA sequences (sequence-specific)Positive acting - activatorsNegative acting - repressorsRegulatory DNA sequencespresent sequence-specificsurface features on the DNAhelixGene regulatory proteinsrecognize these features atthe major groove and formvarious types of contacts(H-bonds, ionic bonds,hydrophobic interactions)usually without disrupting thebase pairing H-bonds.Regulatory proteinstypically form ~20contacts with theirtarget sequence.• High specificityGene regulatory proteinsDNA-binding Motifs:Stable protein folding patterns responsible for DNA bindingExamples:• Homeodomain• Zinc finger• Leucine zipperDNA-binding proteins are frequently dimers• Increased contacts for binding specificity and affinity• Heterodimers can provide greater diversity for sequencerecognition08_05_binding motifs.jpgHomeodomainZinc finger(clusters)Leucine zipperH-bondingIonic bondingLeucine zipper Zinc fingerHydrophobicinteractionspromotedimerizationPositive chargesstabilize DNA bindingOften arranged in clusters withthe finger loop of each makingcontact with DNAGene regulation in bacteria and virusesCoordinate expression of genes (cistrons) in an operonRegulatory DNA sequence, operator, is located within thepromoterNegative regulation:Tryptophan and Lac repressorsPositive regulation:CAP (cyclic AMP binding protein)08_07_repress.protein.jpgThe tryptophan repressor binds to the operator onlywhen it is also bound to tryptophan (allosteric)Activator proteins enhance expression of genes whosepromoters are marginally functional in binding RNApolymerase on their own (in contrast to the promoter forthe tryptophan operon)An activator’s ability to bind DNA also may be affected byallosteric binding to a second moleculeExample: CAP binds DNA only when bound to cAMP08_09_lac operon.jpgThe lac operon is controlled bypositive and negative regulationOnce again, everything ismore complex ineukaryotes.RNA polymerase IIrequires several generaltranscription factors tofind a promoter. The firstand most important isTFIID, a protein complexthat contains the TATAbinding protein.Transcriptional regulation in eukaryotes differs from prokaryotes1. RNA polymerase:• Prokaryotes have a single type• Eukaryotes have three typesRNA pol I: most rRNA genesRNA pol II: protein-encoding genes (makes mRNA)RNA pol III: tRNA, 5S rRNA, small structural RNA genes2. Initiation:• Prokaryotic RNA pol can initiate without helper proteins• Eukaryotic RNA pols require general transcription factors3. Distance between promoter and regulatory protein binding sites:• Prokaryotic regulatory proteins generally bind within the promoter• Eukaryotic regulatory proteins can influence initiation from a greatdistance (>1,000 bp), and may even be downstream of promoter4. Chromatin structure of eukaryotic chromosomes08_24_chromatin.state.jpgStable patterns of gene expression can be transmittedPropagation of chromatin structureGene regulatory proteins can affect local chromatin structureRecruitment of chromatin modifying enzymes/complexes08_13_gene.activation.jpgEukaryotic gene regulatory proteins caninfluence transcription initiation from a distanceEnhancers can be upstream or downstream, and still workif flipped 180˚Multiple DNA-binding proteins can be involved in gene regulationCombinatorial controlExpression is not only on/off, but also where and whenA single regulatory protein can coordinatethe expression of many different genesLike glucocorticoid receptor, thetranscriptional activator MyoD caninfluence the expression of manygenes. When MyoD is introduced intofibroblasts (cells from skin connectivetissue), it is sufficient to causedifferentiation of the cells to formmuscle cells.Differences in gene expression arethe basis for cell differentiation08_22_cell.types.jpgCombinations of even a fewgene regulatory proteins canyield many different cell typesStable patterns of gene expression can be transmittedPositive feedback loop (latch)Transcriptional Network MotifsFeed-forward loop and signal duration07_37_Protein.produc.jpgNearly all steps intranscription andtranslation can beregulated to changelevels of a protein in thecell.Useful text questions8-1, 2, 3, 4, 5, 6,