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Download Chapter 16 Gene Regulation Levels of Gene Regulation Bacterial
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Gene Regulation • Unicellular flexibility – Genes turned on and off in response to environment Chapter 16 Control of Gene Expression (Part 2) • Multicellular specialization – Genes for one cell type are not expressed in other cell types Levels of Gene Regulation • • • • • • Gene Structure Transcription mRNA processing Regulation of mRNA stability Translation Post translational protein modification Figure 16.1 Genes vs. Regulatory Elements • Structural genes: – Metabolism, structure, biosynthesis • Regulatory genes: – Affect transcription or translation – DNA binding proteins • Regulatory elements: – Not transcribed – Affect gene expression Bacterial Gene Regulation • Functionally related genes often clustered • Can be transcribed together on same mRNA • Operon: Operon: Group of bacterial structural genes that are transcribed together. – includes promoters and regulatory elements 1 Modes of Transcriptional Control • Negative – Regulatory protein acts as repressor – Bind to DNA and inhibits transcription • Positive – Regulatory protein acts as activator – Binds to DNA and stimulates transcription 2 Classes of Operon • Inducible – Transcription is normally OFF – Modulator turns transcription ON • Repressible – Transcription is normally ON – Modulator turns transcription OFF Figure 16.6 An Example: Example The lac Operon of E. coli • Involved in lactose metabolism in E. coli • Lactose: – Disaccharide – Doesn’t diffuse across membrane easily • Enzymes: – Β-Galactosidase – Permease – Transacetylase F’ Cells • Cells containing an F plasmid with some bacterial genes. Figure 8.16 2 Partial Diploids Partial Diploids will come in really handy for studying gene expression! (Chapter 16) • Conjugation between an F’ Cell and an Fcell can result in cells with 2 copies of some genes • These are called Partial Diploids or merozygotes Genotypes of Partial Diploids lac Mutations • Partial Diploid strains of E. coli: • Bacterial Chromosome / Plasmid – 2 copies of lac operon – Bacterial chromosome – Plasmid • Examples: • Cis acting mutations: – Control expression of genes on the same piece of DNA only • Trans acting mutations: – lacZ- lacY+ / lacZ+ lacY– Structural mutation of lacZ gene on bacterial chromosome – Structural mutation of lacY gene on plasmid – Control expression of genes on other DNA molecules lacI+ lacZ- / lacI- lacZ+ Figure 16.10 Chromosome Plasmid Chromosome Plasmid 3 Figure 16.11 Types of Mutations I lacIs lacZ+ / lacI+ lacZ+ • Structural gene Mutations – Affect structure of enzymes, not regulation – The wild type is Trans Dominant • Regulator gene Mutations – Constitutive: lac enzymes produced constantly (in regular E. coli) – In partial diploids, lacI+ is Trans Dominant lacIs encodes a superrepressor Figure 16.12 Types of Mutations II Constitutive! • Operator mutations – lacOc indicates a mutation in the DNA sequence of the operator – Repressor cannot bind to operator – lacOc is cis dominant and constitutive Figure 16.11 Figure 16.11 Constitutive! Cis acting! 4 Figure 16.11 Types of Mutations III • Promoter mutations Cis acting! – lacP- indicates a mutation in the DNA sequence of the promoter – RNA polymerase cannot bind to promoter – lacP- is cis dominant lacI+ lacP- lacOc lacZ+ lacY- / lacI- lacP+ lacO+ lacZ- lacY+ • What is the ENZYMATIC ACTIVITY? ACTIVITY • Lactose Absent Lactose Present • B-Gal • ? B-Gal ? Permease ? Permease ? • Use “-” for no activity and “+” for activity lacI+ lacP- lacOc lacZ+ lacY- / lacI- lacP+ lacO+ lacZ- lacY+ • What is the ENZYMATIC ACTIVITY? ACTIVITY • Lactose Absent Lactose Present • A) B) C) D) B-Gal Permease + + + B-Gal + + + Permease + + + - lacI+ lacZ- / lacI- lacZ+ Figure 16.9 Chromosome Plasmid Chromosome Plasmid 5 Catabolite Repression CAP and cAMP • Glucose is the preferred food source for E. coli • Catabolite Acitvator Protein • When glucose is available: • Cyclic AMP (adenosine-3’,5’-cyclic monophosphate) – Genes for metabolism of other sugars are repressed – Catabolite Repression – Binds to DNA upstream of lac promoter – RNA polymerase won’t bind efficiently to lac promoter unless CAP is first bound to DNA – CAP can’t bind to DNA without cAMP – Concentration of cAMP inversely proportional to glucose concentration Figure 16.12 trp Operon • Controls biosynthesis of tryptophan • Negative repressible operon This is POSITIVE CONTROL because CAP is an ACTIVATOR Figure 16.14 Attenuation • Another form of transcriptional control for the trp operon. • Transcription is initiated but terminates prematurely. 6 Figure 16.14 Figure 16.14 Look Familiar? Rho-independent Termination Rho-independent Termination 1) Two inverted repeats in the DNA sequence are transcribed 2) A string of ~6 Adenines follows the second inverted repeat 3) The inverted repeats form a hairpin structure pausing the polymerase 4) The A-U bonds break and the RNA molecule separates from the template Figure 16.14 Antisense RNA • RNA regulator of gene expression • Antisense RNA Check out the online animation for the lac operon and attenuation – Small RNA molecules complementary to certain sequences on mRNAs. – Inhibit translation • Example: ompF gene of E. coli – Important in cellular osmoregulation – Increased osmolarity turns on micF – micF produces Antisense RNA 7 Figure 16.17 Figure 16.17 Ribosome cannot bind Eukaryotic Gene Regulation • • • • No operons in Eukaryotes Chromatin affects gene expression Activators are more common Many mechanisms at many levels Eukaryotic Gene Regulation Eukaryotic Gene Regulation • • • • • • Gene Structure Transcription mRNA processing Regulation of mRNA stability Translation Post translational protein modification Gene Regulation: Gene Structure (Chromatin) • DNAaseI Hypersensitivity – DNAase I digests DNA – Doesn’t work when DNA tightly bound to histones • Transcriptionally active genes – DNAaseI hypersensitive sites • Regions near transcriptionally active genes where DNA configuration is more open • DNA binding proteins? 8 Gene Regulation: Gene Structure (Chromatin) cont. • Histone acetylation – Facilitates transcription • DNA methylation – Cytosine bases methylated – Associated with transcription repression – CpG islands: islands …GC… …CG… Gene Regulation: Transcriptional Control • Transcriptional activators – Stabilize basal transcription apparatus (BTA) – Often interact with BTA through coactivators – Stimulate transcription • Repressors – May bind to regulatory promoter – May bind to silencers Figure 16.23 ENHANCERS AND INSULATORS • Enhancers affect transcription at distant promoters – Alpha chain of Tcell receptor: enhancer is 69,000 bp downstream of promoter – Enhancers can stimulate any promoter in its vicinity • Insulators (boundary elements) limit the effect of enhancers RESPONSE ELEMENTS • Response elements – DNA regulatory elements which are bound by transcriptional activator proteins. • Example: Metallothionein – Response elements to heavy metals • Eukaryotic Genes may be activated by several different response elements Multiple Response Elements (MREs) allow the same gene to be activated by different stimuli. 9 Response elements to a particular stimulus can be associated with multiple genes, allowing a single stimulus to activate multiple genes. Gene Regulation: Messenger RNA Processing • Alternative Splicing: Splicing – SR Proteins: often regulate splicing – Example: T-antigen gene of mammalian virus SV40 • Splicing Factor 2 (SF2 SF2) is a type of SR Protein – Another Example: Sex determination in Drosophila. Gene Regulation: Translation and Posttranslational Control • Availability of Translational Apparatus: Apparatus – Ribosomes, aminoacyl tRNAs, initiation factors, elongation factors. – Less available: slower translation. • Proteins binding to 5’ UTR • Posttranslational modification – Trimming, acetylation, addition of phosphates, carboxyl groups, etc. Gene Regulation: RNA Stability Variation in mRNA Stability Variation in Protein Production • Stability of mRNA affected by: – 5’ Cap – Poly (A) tail – 5’ UTR – Coding region – 3’ UTR RNA Interference (RNA Silencing) • Double stranded RNA initiates a cascade that degrades complementary mRNA. • May have evolved as a defense against double stranded RNA viruses. • Very handy for artificially regulating gene expression – Model organisms – Genetically engineered organisms 10 11