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Gene Regulation III Reminder • Exam Friday Review • • • • DNA ➞ RNA ➞ protein Every cell in body has same DNA. Not all cells make all proteins What turns genes on and off? !2 Prokaryotes •small (1000x) •no nucleus •no membranebound organelles •1 main chromosome •some plasmid •about 4000 genes •genes in operons Promoter Operator gene A gene B gene C Lac Operon - Negative Control lac i Promoter Operator Lac z Lac y Lac a repressor !4 Lac Operon - Inducible lac i RNA PolymerPromoter Operator ase Lac z BG- Lac y Lac a P TA Lac Operon - Positive Control lac i RNA CAP PolymerPromoter Operator ase cAMP Lac z BG- Lac y Lac a P TA !6 Trp Operon trp r inactive repressor RNA Polymer- P ase O trpE trpD trpC trpB trpA E- D C B A Trp Operon - Repressible trp r RNA PolymerP ase tryptophan O trpE trpD trpC trpB trpA Transcription/Translation Simultaneous DNA trp E • Prokaryotes no nucleus trp D trp C trp B trp A Transcription mRNA Translation Attenuation/Leader mRNA 4 Low trp in env. •Translation of 1 slow •2-3 loop forms •transcription of complete mRNA 1 4 3 1 trpE trpD trpC trpB trpA 3 2 L 2 High trp in env. •Translation of 1 fast •3-4 termination hairpin forms •inhibits further transcription Eukaryotes • have nucleus • have membranebound organelles • cells larger • DNA in nucleus • DNA linear • Transcription & translation separate places (nucleus vs ribosome) • More chromosomes • More genes (20,000-50,000) No Operons • Related Genes Separate Hemoglobin Beta chain Alpha chain Beta chain Alpha chain Beta Gene Chromosome 11 Alpha Gene Chromosome 16 Have Introns and Exons Alpha Hemoglobin Gene E-I E-II Introns E-III DNA Packing • DNA Wraps Around Proteins • Folds several times Nucleosome DNA wrapped 2x around 8 histones Spacer Histones (+ Charge) DNA - 2 nm Nucleosome - 11 nm Solenoid Ring of nucleosomes 30 nm Chromatin and Chromosome Chromosome (Division) Highly Folded 700 nm Solenoid 30 nm Chromatin (Interphase) Looped Solenoids 300 nm Chromosome Territories Each chromosome has own region in nucleus Heterochromatin vs Euchromatin • dark-staining • tightly coiled DNA • genetically inactive • • • • Found centromere telomeres entire Y Inactive X (Barr body) • light staining • relatively uncoiled • genetically active DNA Methylation addition of -CH3 to base or sugar Cytosine Cytosine NH2 NH2 CH3 O O DNA closed, inactive Chromatin Remodeling • alteration of DNA association with histones • necessary for gene transcription Acetylation addition of acetyl group to histone NH3 + CH2 histone acetyltransferase HAT CH2 CH2 Lysine + charge CH2 CH2 NH -CH2-CH3 CH2 histone deacetylase HDAC CH2 CH2 Lysine no charge less attraction to DNA Epigenetics • Changes in phenotype or gene expression that do not involve changes in DNA sequences • Caused by differences in methylation and acetylation Gene Organization DN E P Gene S Promoter - DNA region - RNA polymerase attaches - upstream Enhancer - DNA region - increase transcription upstream or downstream or intron Silencer - DNA region - decrease transcription upstream or downstream or intron Transcription Factors • proteins that bind to DNA to affect transcription IIB TAFs TBP IIA IIJ IIF IIH IIE Basal (General)Transcription Factors Attach to promoter Attract RNA Polymerase RNA IIB IIF Polymerase TBP IIA TAFs IIJ IIH Promoter IIE Activators • increase transcription • Some bind to enhancer/promoter to form loop Enhancer IIB TAFs TBP IIA RNA Polymerase IIJ IIF IIE IIH Promoter Transcription Video Activators • others attract enzymes to open chromatin HAT RNA Polymerase Repressors • inhibit transcription • binds to silencer • attract enzymes to close chromatin HDA R Transcription Factor Attachment • environmental signal • binding of another transcription factor Phytochrome •Causes plant responses to light Pr TA Fs II J II frII IIE P Pfr red light Estrogen • female sex hormone Estrogen Inactive Receptor TA FsActive II II J Receptor II IIE Active Receptor Post-Transcriptional Control 1. Alternative Splicing of Exons mG E1 E2 E3 E5 E6 AAAAAA... neuropeptide Brain E1 mRNA 1o Transcript E2 E3 E4 E5 thyroid mRNA Processed mG E1 E2 E3 E4 Transcript AAAAAA... calcitonin E6 Post-Transcriptional Control 2. RNA Interference (RNAi) • Process that uses small RNA molecules to regulate gene expression • 2 sources of RNA ‣ cell’s own genes - MicroRNA (miRNA) ‣ viruses and transposons - Small Interfering RNA (siRNA) MicroRNA Typical Gene mRN Microgene MicroRNA DNA Protein binds to Blocks Transcription MicroRNA Dicer RISC RNA Interference http://www.youtube.com/watch?v=_-9pROnSD-A RNA Interference siRNA or miRNA RNA Induced Silencing Complex RISC Complex binds to mRNA and stops translation RNA Induced Transcriptional Silencing RITS complex Complex binds to DNA histones and blocks transcription Post-Transcriptional Control 2. RNA Interference (RNAi) • Process that uses small (20-25 bases) RNA molecules to regulate gene expression • 2 sources of RNA ‣ cell’s own genes - MicroRNA (miRNA) ‣ viruses and transposons - Small Interfering RNA (siRNA) • Process likely evolved to inactivate viral genes and transposons Microgene Discovery scientists trying to genetically engineer petunia to be darker got white patches due to RNA interference MicroRNA Research C. elegans (roundworm) grows fast (3 days) reproduces (3 days) hundreds of offspring •Microgenes found in all eukaryotic organisms •Over 1000 in human genome •analogous process in bacteria Potential Uses iRNA • research into gene function • pharmaceutical use • block expression of abnormal gene • block overexpression of normal gene (viruses, cancer) Activity 18 !42