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AP Biology Discussion Notes Monday 3/13/2017 Goals for Today • Be able to describe regions of DNA and how they are important to gene expression in Bacteria (Prokaryotes) & Eukaryotes • Be able to describe both inducible & repressible operons and explain the difference between the two • Be able to understand & describe how our Transformation Lab works 3/13 Question of the Day Identify the two major processes within Protein Synthesis. Gene Review What cellular machinery is necessary to make proteins? DNA mRNA Protein Transcription: Translation: DNA RNA Polymerase Nucleotides Ribsosome (mRNA) tRNA Amino Acids Gene Expression/Regulation • The Nobel Prize in Physiology or Medicine 1965 was awarded jointly to François Jacob, André Lwoff and Jacques Monod "for their discoveries concerning genetic control of enzyme and virus synthesis". Conducting the Genetic Orchestra CH18 • Prokaryotes and eukaryotes alter gene expression in response to their changing environment 18.1: Bacteria often respond to environmental change by regulating transcription • Natural selection has favored bacteria that produce only the products needed by that cell • A cell can regulate the production of enzymes by feedback inhibition or by gene regulation • Gene expression in bacteria is controlled by the operon model Upstream PROG Downstream Upstream OPERON Promoter – helps bind RNA Polymerase Repressor (Protein)–stops transcription, binds to OPERATOR Operator – ON/OFF switch, place where repressor binds – Control operation Genes – Code for protein Down stream Regulator Gene – codes for repressor protein Let’s Draw it! Operons: The Basic Concept • A cluster of functionally related genes can be under coordinated control by a single “on-off switch” • The regulatory “switch” is a segment of DNA called an operator usually positioned within the promoter • An operon is the entire stretch of DNA that includes the operator, the promoter, and the genes that they control • The operon can be switched off by a protein repressor • The repressor prevents gene transcription by binding to the operator and blocking RNA polymerase • The repressor is the product of a separate regulatory gene Repressible and Inducible Operons An inducible operon is one that is usually ____; a molecule called an inducer inactivates the repressor and turns on transcription What is the “inducer” in the Lac Operon? What is the INDUCER in the lac Operon? lac operon lacI DNA lacZ lacY lacA Permease Transacetylase RNA polymerase 3 mRNA 5 mRNA 5 -Galactosidase Protein Allolactose (inducer) Inactive repressor (b) Lactose present, repressor inactive, operon on Figure 18.4a Regulatory gene DNA Promoter Operator lacI lacZ No RNA made 3 mRNA 5 Protein RNA polymerase Active repressor (a) Lactose absent, repressor active, operon off Positive Gene Regulation • Some operons are also subject to positive control through a stimulatory protein, such as catabolite activator protein (CAP), an activator of transcription • When glucose (a preferred food source of E. coli) is scarce, CAP is activated by binding with cyclic AMP (cAMP) • Activated CAP attaches to the promoter of the lac operon and increases the affinity of RNA polymerase, thus accelerating transcription Positive Gene Regulation • When glucose levels increase, CAP detaches from the lac operon, and transcription returns to a normal rate • CAP helps regulate other operons that encode enzymes used in catabolic pathways Figure 18.5a Promoter DNA lacI lacZ CAP-binding site cAMP Operator RNA polymerase Active binds and transcribes CAP Inactive CAP Allolactose Inactive lac repressor (a) Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized Figure 18.5b Promoter DNA lacI CAP-binding site lacZ Operator RNA polymerase less likely to bind Inactive CAP Inactive lac repressor (b) Lactose present, glucose present (cAMP level low): little lac mRNA synthesized Questions? Repressible and Inducible Operons: • A repressible operon is one that is usually ____; binding of a repressor to the operator shuts off transcription • The trp operon is a repressible operon trp OPERON • The repressor can be in an active or inactive form, depending on the presence of other molecules • The repressor is active only in the presence of its corepressor tryptophan; – A corepressor is a molecule that cooperates with a repressor protein to switch an operon off • thus the trp operon is turned off (repressed) if tryptophan levels are high trp OPERON • By default the trp operon is on; and the genes for tryptophan synthesis are transcribed • When tryptophan is present, it binds to the trp repressor protein, which turns the operon off trp OPERON • E. coli can synthesize the amino acid tryptophan • Think of these questions and think about energy and natural selection: – When would E. coli “want” to do this? – When would E. coli “not want” to do this? Figure 18.3a trp operon Promoter Promoter Genes of operon DNA trpR Regulatory gene mRNA trpE 3 Operator RNA Start codon polymerase mRNA 5 trpD trpC trpB trpA C B A Stop codon 5 E Protein Inactive repressor D Polypeptide subunits that make up enzymes for tryptophan synthesis (a) Tryptophan absent, repressor inactive, operon on Figure 18.3b-1 DNA mRNA Protein Active repressor Tryptophan (corepressor) (b) Tryptophan present, repressor active, operon off Figure 18.3b-2 DNA No RNA made mRNA Protein Active repressor Tryptophan (corepressor) (b) Tryptophan present, repressor active, operon off Figure 18.2 Precursor Feedback inhibition trpE gene Enzyme 1 trpD gene Enzyme 2 Regulation of gene expression trpC gene trpB gene Enzyme 3 trpA gene Tryptophan (a) Regulation of enzyme activity (b) Regulation of enzyme production Figure 18.3 trp operon Promoter Promoter Genes of operon DNA trpE trpR trpD trpC trpB trpA C B A Operator Regulatory gene 3 RNA polymerase Start codon Stop codon mRNA 5 mRNA 5 E Protein Inactive repressor D Polypeptide subunits that make up enzymes for tryptophan synthesis (a) Tryptophan absent, repressor inactive, operon on DNA No RNA made mRNA Protein Active repressor Tryptophan (corepressor) (b) Tryptophan present, repressor active, operon off Repressible and Inducible Operons: • A repressible operon is one that is usually ____ • An inducible operon is one that is usually ____ • The trp operon is a(n) ____________ operon • The Lac operon is a(n) ____________ operon Conducting the Genetic Orchestra CH18 • Prokaryotes and eukaryotes alter gene expression in response to their changing environment • In multicellular eukaryotes, gene expression regulates development and is responsible for differences in cell types Figure 18.1 Eukaryotic gene expression: regulated at many stages • All organisms must regulate which genes are expressed at any given time • In multicellular organisms regulation of gene expression is essential for cell specialization Figure 11.22 Interdigital tissue Cells undergoing apoptosis Space between 1 mm digits Differential Gene Expression • Almost all the cells in an organism are genetically identical • Differences between cell types result from differential gene expression, – the expression of different genes by cells with the same genome • Abnormalities in gene expression can lead to diseases including cancer • Gene expression is regulated at many stages Figure 18.6 Signal NUCLEUS Chromatin DNA Chromatin modification: DNA unpacking involving histone acetylation and DNA demethylation Gene available for transcription Gene Transcription RNA Exon Primary transcript Intron RNA processing Cap Tail mRNA in nucleus Transport to cytoplasm CYTOPLASM mRNA in cytoplasm Degradation of mRNA Translation Polypeptide Protein processing, such as cleavage and chemical modification Degradation of protein Active protein Transport to cellular destination Cellular function (such as enzymatic activity, structural support) Figure 18.6a Signal NUCLEUS Chromatin DNA Chromatin modification: DNA unpacking involving histone acetylation and DNA demethylation Gene available for transcription Gene Transcription RNA Exon Primary transcript Intron RNA processing Cap Tail mRNA in nucleus Transport to cytoplasm CYTOPLASM Figure 18.6b CYTOPLASM mRNA in cytoplasm Degradation of mRNA Translation Polypeptide Protein processing, such as cleavage and chemical modification Degradation of protein Active protein Transport to cellular destination Cellular function (such as enzymatic activity, structural support) DNA Methylation • DNA methylation, the addition of methyl groups to certain bases in DNA, is associated with reduced transcription in some species • DNA methylation can cause long-term inactivation of genes in cellular differentiation DNA Methylation • In genomic imprinting, methylation regulates expression of either the maternal or paternal alleles of certain genes at the start of development Histone Modifications • In histone acetylation, acetyl groups are attached to positively charged lysines in histone tails • This loosens chromatin structure, thereby promoting the initiation of transcription • The addition of methyl groups (methylation) can condense chromatin; the addition of phosphate groups (phosphorylation) next to a methylated amino acid can loosen chromatin Figure 18.7 Histone tails Amino acids available for chemical modification DNA double helix Nucleosome (end view) (a) Histone tails protrude outward from a nucleosome Acetylated histones Unacetylated histones (b) Acetylation of histone tails promotes loose chromatin structure that permits transcription Operon Practice • If you can draw it you understand it