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GENE EXPRESSION AND UNNATURAL MANIPULATION What would be the outcome of not regulating of gene expression? 5/23/2017 2 AP Ch 18 Regulation of Gene Expression 5/23/2017 3 Why don’t all cells in your body look/work the same if all are products of mitosis from the zygote? How can bacterial cells differentiate/evolve without sex or meiosis? 5/23/2017 5 How can bacterial cells differentiate/evolve without sex or meiosis? • mutations occur every 107 cells, E.coli make 2x1010 a day, ∴ 2000 mutants a day = a lot of variation 5/23/2017 6 Related bacteria terms (skip for now) • Transformation – alter a bacteria’s genotype by the uptake of (plasmid) DNA • Plasmid – small, circular, self-replicating piece of DNA – R plasmids – special – genes inserted • Transduction – transfer of bacterial genes via phages • Conjugation – direct transfer of genetic info. between 2 joined bacteria (pilus), like sex • Transposons – “jumping genes” genes that move or get duplicated into other parts of the genome 5/23/2017 7 Gene control (in bacteria) • Operons- group of genes that controls expression, Starts with the promoter, RNA polymerase binds • operator turns transcription on, mRNA gets made – repressor – protein that can stop transcription by binding to the operator, there are also corepressors that help – inducer – activates by inactivating the repressor (binds) • ex. lac operon → turns on when lactose is present because allolactose binds to the repressor, makes genes that digest lactose 5/23/2017 8 OPERONS •Repressible (trp) vs inducible (lac) enzymes • Negative vs positive gene regulation 5/23/2017 9 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 Eukaryotic Genomes 5/23/2017 11 Signal NUCLEUS Chromatin DNA Stages in eukaryotic genome expression 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) Gene Expression Control • 20% of genes in humans expressed at a given time • control occurs at any stage from replication to post translation 5/23/2017 13 Major Methods of Gene Regulation How are color groups related? 1. Histone Acetylation: promotes transcription b/c it opens tightly packed nucleosomes, giving transcription proteins easier access (–COCH3 ) 2. DNA methylation: add –CH3 groups to DNA, often shuts genes off 3. Control elements before the coded DNA that regulate transcription = transcription factors 4. Splicing of RNA by spliceosomes 5. Non-coding RNAs: siRNAs (small interfering RNA), miRNAs (micro RNA) degrade transcripts or block translation 6. Protein degradation 5/23/2017 14 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 Transcription activators Activators Promoter DNA Enhancer Distal control element TATA box Gene Transcription activators Promoter Activators DNA Enhancer Distal control element Gene TATA box General transcription factors DNAbending protein Group of mediator proteins Figure 18.10-3 Promoter Activators DNA Enhancer Distal control element Gene TATA box General transcription factors DNAbending protein Group of mediator proteins RNA polymerase II RNA polymerase II Transcription initiation complex RNA synthesis Enhancer Expression of different genes in different cell types Control elements Promoter Albumin gene Crystallin gene LENS CELL NUCLEUS LIVER CELL NUCLEUS Available activators Available activators Albumin gene not expressed Albumin gene expressed Crystallin gene not expressed (a) Liver cell Crystallin gene expressed (b) Lens cell Splicing: Regulation Method # 4 Enhancer (distal control elements) DNA Upstream Proximal control elements Transcription start site Exon Intron Exon Intron Downstream Poly-A signal Intron Exon Exon Cleaved 3 end of primary RNA processing transcript Promoter Transcription Exon Primary RNA transcript 5 (pre-mRNA) Poly-A Transcription signal sequence termination region Intron Exon Intron RNA Coding segment mRNA G P AAA AAA P P 5 Cap 5 UTR Start Stop codon codon 3 UTR Poly-A tail 3 RNA degradation: Regulation Method # 5 Regulation and gene expression by miRNAs (micro RNA) 5/23/2017 21 RNAi– A form of siRNA (small interfering RNA) • Used to treat various genetically-based disorders – Recall bio 1 Nova: Science Now video 5/23/2017 22 Cell Differentiation – How does a cell know what job it will do? • 250 different cell types, all from one “stem” cell, HOW? • Differentiation = cell becomes specialized in structure or function • Morphogenesis =organisms shape is established • mice, C. elegans & fruit flies etc.. used to study development: goal is to find a “cells lineage” 5/23/2017 23 3 Factors that influence early development 1) Influence of the egg’s cytoplasm • cytosol is distributed unevenly, causes differences in the new cells • axis of the developing egg cell • the egg’s RNA 2) embryonic induction - chemical signals from neighboring cells signal change 5/23/2017 24 Sources of developmental info for the embryo 5/23/2017 25 • 3) Homeotic Genes • hox genes, turn genes on & off • 180 base segment, called a homeobox that is consistent across species, evolution… p.370 and p.445 • Apoptosis - programmed cell death, all cells are destined to die – -why? Essential for proper development, ex. webbed feet, when cells go "bad" 5/23/2017 26 5/23/2017 27 Why did this occur? 5/23/2017 28 Cancer: from expression of cell cycle genes • Oncogene- cancer causing, 1 copy is bad = cancer • Tumor suppressor – prevent uncontrolled cell growth, both copies must be faulty p53- fix DNA or shut bad DNA off, 5-7 things must happen for cancer to occur 5/23/2017 29 Figure 18.24 Two different transcription pathways can result in cancerous formations MUTATION 1 Growth factor Ras 3 G protein GTP Ras P P P P P P 2 Protein kinases Hyperactive Ras protein (product of oncogene) issues signals on its own. GTP MUTATION 3 Active form of p53 UV light 2 Receptor 4 Protein kinases (phosphorylation cascade) 1 DNA damage in genome Defective or missing transcription factor, such as p53, cannot activate transcription. DNA NUCLEUS 5 Transcription factor (activator) Protein that inhibits the cell cycle DNA Gene expression (b) Cell cycle–inhibiting pathway Protein that stimulates the cell cycle EFFECTS OF MUTATIONS Protein overexpressed Protein absent (a) Cell cycle–stimulating pathway Cell cycle overstimulated (c) Effects of mutations Increased cell division Cell cycle not inhibited Explain one of the following Turning proto-oncogenes into oncogenes Proto-oncogene DNA Translocation or transposition: gene moved to new locus, under new controls Gene amplification: multiple copies of the gene New promoter Normal growthstimulating protein in excess Point mutation: within a control within element the gene Oncogene Normal growth-stimulating protein in excess Normal growthstimulating protein in excess Oncogene Hyperactive or degradationresistant protein Transcription Chromatin modification • Genes in highly compacted chromatin are generally not transcribed. • Histone acetylation seems to loosen chromatin structure, enhancing transcription. • DNA methylation generally reduces transcription. • Regulation of transcription initiation: DNA control elements in enhancers bind specific transcription factors. Bending of the DNA enables activators to contact proteins at the promoter, initiating transcription. • Coordinate regulation: Enhancer for Enhancer for liver-specific genes lens-specific genes Chromatin modification Transcription RNA processing RNA processing • Alternative RNA splicing: Primary RNA transcript mRNA degradation Translation Protein processing and degradation mRNA or Translation • Initiation of translation can be controlled via regulation of initiation factors. mRNA degradation • Each mRNA has a characteristic life span, determined in part by sequences in the 5 and 3 UTRs. SUMMARY Protein processing and degradation • Protein processing and degradation by proteasomes are subject to regulation. Quick Quiz Quick Quiz 1. Name four ways in which genes can be regulated. Quick Quiz 1. Name four ways in which genes can be regulated. 2. What chemical group would bind up a strand of DNA inhibiting transcription? Quick Quiz 1. Name four ways in which genes can be regulated. 2. What chemical group would bind up a strand of DNA inhibiting transcription? 3. What would be the effect of reordering hox genes? Quick Quiz 1. Name four ways in which genes can be regulated. 2. What chemical group would bind up a strand of DNA inhibiting transcription? 3. What would be the effect of reordering hox genes? 4. Name four structures that participate in transcription regulation in eukaryotes. Quick Quiz 1. Name four ways in which genes can be regulated. 2. What chemical group would bind up a strand of DNA inhibiting transcription? 3. What would be the effect of reordering hox genes? 4. Name four structures that participate in transcription regulation in eukaryotes. 5. What does the operon include? 6. How can RNAi be used to fight disease? Quick Quiz 1. Name four ways in which genes can be regulated. Slide 14 2. What chemical group would bind up a strand of DNA inhibiting transcription? 14-15 3. What would be the effect of reordering hox genes? 28 4. Name four structures that participate in transcription regulation in eukaryotes. 18 5. What does the operon include? 9 6. How can RNAi be used to fight disease? vid