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Download Bio1A Unit 2-7 Gene Expression Pt 1 Notes File
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Mutation: by function • Mutants are almost always “loss of function” whatever you mutated doesn’t work. There are far more ways for things to go wrong than right. The alternative is a “gain of function” mutation. • Think of mutations in terms of an enzyme. substrate enzyme Product Ex: Pigment = Red Mutant substrate No Product Not red • mutations are just in DNA sequence. There is more non-coding DNA than coding in the (human) genome. • We will consider regulatory elements which are genes AND promoter elements Control of Gene expression • Gene is “expressed” when: – It is transcribed – RNA is translated – Protein is active • organisms respond to environment by controlling the expression of their genes • Multi-cellular organisms express different genes in different cell types Lactose metabolism in bacteria • Bacteria can digest lactose - requires proteins / enzymes. i.e. – β-galactosidase • Bacteria will not produce lactose metabolic enzyme unless needed Lactose present • If lactose is absent enzyme are not made in order to conserve energy No β-gal • Bacteria prefer Glucose (fewer enzymatic steps = fewer enzymes = more efficient) • When glucose is present lactose enzymes not make Efficient = growth advantage Inefficient bacteria don’t die, they are just become minority (subpopulation) Operons – Bacteria, not Eukaryotes • entire stretch of DNA that includes the operator, the promoter, and the genes that they control • cluster of (related) genes can be under coordinated control by a single on-off “switch” (Operator) • operator usually positioned within the promoter Promoter Operator Multiple SD - start/stops Lac Repressor • regulatory gene / separate protein under another promoter distinct from the operon • repressor binds to operator & prevents transcription by blocking RNA polymerase Lac Operon is inducible • By itself, the lac repressor is active and switches the lac operon off • A molecule called an inducer inactivates the repressor to turn the lac operon on • The inducer, lactose, inactivates lac repressor to turn lac operon on Experimental Analysis of Gene Regulation Tools Specific to Lactose Regulation Substrate analogs - A compound that “looks” like the native (original) substrate IPTG Looks like allolactose Inducer of LacI But not degraded X-gal Indicator Looks like lactose (Not an inducer) Recognized by β-gal Used to detect: the expression (presence) of β-gal = if lac operon is on Produces blue precipitate Lac Operon Discovery Genetics: find genes & other regulatory elements Mutagenesis Experiment X-gal IPTG X-gal NO IPTG NO inducer LacI Repressor binds Block RNA Pol No transcription No β-gal doesn’t digest X-gal Mutagenesis X-gal NO IPTG + inducer LacI Repressor inactivated Transcription β-gal made digests X-gal Blue Lac I mutant (loss of function) NO inducer LacI Repressor binds Block RNA Pol = No transcrip. No β-gal doesn’t digest X-gal Transcription β-gal made digests X-gal Blue Generically: In a mutagenesis experiment • Mutations in a negative regulator will result in a constitutive mutant (always on, even when it would normally be off) • Mutations in a positive regulator will result in an uninducible mutant (always off, even when it would normally be on) Glucose regulates lac operon Glucose • Best energy source directly enters glycolysis • All other compounds require energy to modify • E. Coli will not induce lac operon if glucose is present. Even when lactose inducer is present • When Glucose is present cAMP is not produced Glucose no Camp No CAP binding No transcription White No Glucose cAMP cAMP bind CAP CAP binds CAP binding site RNA polymerase attracted Gene expression turned on = β-gal made β-gal digests x-gal Blue Repressible and Inducible Operons: Two Types of Negative Gene Regulation • An inducible operon is one that is usually off; an inducer inactivates the repressor (or activates and activator) and turns on transcription • A repressible operon is one that is usually on; corepressor gives binding of a repressor to the operator shuts off transcription • The lac operon is an inducible operon • The trp operon is a repressible operon • The repressor can be in an active or inactive form, depending on the presence of other molecules • A corepressor is a molecule that cooperates with a repressor protein to switch an operon off • Operator & repressor, activator/enhancer, inducer, corepressor Terminology Repressors: • Protein - ↓ gene expression • binds to upstream elements to block RNA polymerase / transcription Activtators: (a.k.a. – enhancer) • Protein -↑ gene expression • binds to upstream elements to aid RNA polymerase binding / transcription Inducers, Corepressor, Coactivators (prok. vs euk.) – small molecules Inducers: • non-protein, small molecules - ↑ gene expression (either by binding and removing repressors or binding activators to cause them to bind their activator binding site) Corepressors: • prokaryotes: non-protein, small molecules - ↓ gene expression (either by removing activators or causing repressor to bind) • Eukaryotes: protein that bind repressors to ↓ gene expression but don’t bind DNA themselves Coactivtators: (only eukaryotes) • Protein that binds activator to ↑ gene expression , but doesn’t bind DNA itself (only eukaryotes) • No analogous term in prokaryotes and so the terms corepressor and coactivator are blurred for prokaryotes and eukaryotes Eukaryotic Genes have multiple enhancers for transcription factors vs Lac operon only has room next to promoter E1 E2 E3 E4 Far away from promoter TATA While no operon system in eukaryotes, coordinate regulation is accomplished by genes having the same enhancer elements Promoters typically have negligible basal activity.Can be regulated by multiple enhancers for complex decisions that vary due to conditions such as: • Cell types • Specific time in development • Specific physiological conditions