Gene Regulation Chapter 14 Learning Objective 1 • Why do bacterial and eukaryotic cells have different mechanisms of gene regulation? Prokaryotes • Bacterial cells • • • grow rapidly have a short life span Transcriptional-level control • usually regulates gene expression Eukaryotic Cells • Have long life span • • One gene • • respond to many different stimuli may be regulated in different ways Transcriptional-level control • and control at other levels of gene expression KEY CONCEPTS • Cells can synthesize thousands of proteins • • but not all proteins are required in all cells Cells regulate which parts of the genome will be expressed, and when Learning Objective 2 • What is an operon? • What are the functions of the operator and promoter regions? Operon • A gene complex • • • structural genes with related functions controlled by closely linked DNA sequences Regulated genes in bacteria • are organized into operons Promoter Region • Each operon has a promoter region • • upstream from protein-coding regions where RNA polymerase binds to DNA before transcription Operator (1) • Regulatory switch for transcriptional-level control of operon • Repressor protein • • binds to operator sequence prevents transcription Operator (2) • RNA polymerase • • • bound to promoter is blocked from transcribing structural genes If repressor is not bound to operator • transcription proceeds Learning Objective 3 • What is the difference between inducible, repressible, and constitutive genes? Inducible Genes (1) • An inducible operon • • • such as lac operon is normally turned off Repressor protein • • is synthesized in active form binds to operator Inducible Genes (2) • If lactose is present • • • • is converted to allolactose (inducer) binds to repressor protein changes repressor’s shape Altered repressor • • cannot bind to operator operon is transcribed The lac Operon lac operon Repressor gene Promoter Operator lac Z lac Y lac A DNA Repressor protein Transcription mRNA Ribosome Translation Fig. 14-2a, p. 307 Repressor gene mRNA lac operon Promoter Operator lac Z lac Y RNA polymerase lac A Transcription mRNA Translation Inducer (allolactose) Repressor protein (inactive) Transacetylase Lactose permease β-galactosidase Enzymes for lactose metabolism Fig. 14-2b, p. 307 Repressible Genes (1) • A repressible operon (trp operon) • • Repressor protein • • • is normally turned on is synthesized in inactive form cannot bind to operator A metabolite (metabolic end product) • acts as corepressor Repressible Genes (2) • With high intracellular corepressor levels • • • corepressor molecule binds to repressor changes repressor’s shape Altered repressor • • binds to operator turns off transcription of operon The trp Operon Repressor gene trp operon Promoter Operator trp E trp D trp C trp B trp A DNA mRNA RNA polymerase Transcription mRNA Translation Repressor protein (inactive) Enzymes of the tryptophan biosynthetic pathway Tryptophan (a) Intracellular tryptophan levels low. Fig. 14-4a, p. 310 Repressor gene trp operon Promoter Operator trp E DNA trp D trp C trp B trp A Active repressor – corepressor complex mRNA Inactive repressor protein Tryptophan (corepressor) (b) Intracellular tryptophan levels high. Fig. 14-4b, p. 310 Constitutive Genes (1) • Are neither inducible nor repressible • • active at all times Regulatory proteins • produced constitutively • catabolite activator protein (CAP) • repressor proteins Constitutive Genes (2) • Regulatory proteins • • recognize and bind to specific base sequences in DNA Activity of constitutive genes • controlled by binding RNA polymerase to promoter regions Learning Objective 4 • What is the difference between positive and negative control? • How do both types of control operate in regulating the lac operon? Negative Control • Repressible and inducible operons are under negative control • When repressor protein binds to operator • transcription of operon is turned off Positive Control (1) • Some inducible operons are under positive control • Activator protein binds to DNA • stimulates transcription of gene Positive Control (2) • CAP activates lac operon • • • To bind to lac operon • • binds to promoter region stimulates transcription by tightly binding RNA polymerase CAP requires cyclic AMP (cAMP) cAMP levels increase • as glucose levels decrease Positive Control Promoter Repressor gene CAPRNA binding polymerase – site binding site Operator lac Z lac Y lac A DNA mRNA RNA polymerase binds poorly CAP (inactive) Allolactose Repressor protein (inactive) (a) Lactose high, glucose high, cAMP low. Fig. 14-5a, p. 311 Promoter Repressor gene CAPRNA binding polymerase – site binding site Operator lac Z lac Y lac A DNA RNA Transcription polymerase binds efficiently mRNA CAP mRNA Translation cAMP Allolactose Repressor protein (inactive) (b) Lactose high, glucose low, cAMP high. Galactoside transacetylase Lactose permease β -galactosidase Enzymes for lactose metabolism Fig. 14-5b, p. 311 Binding CAP DNA cAMP CAP dimer Fig. 14-6, p. 312 Learning Objective 5 • What are the types of posttranscriptional control in bacteria? Posttranscriptional Controls in Bacteria • Translational control • • regulates translation rate of particular mRNA Posttranslational controls • include feedback inhibition of key enzymes in metabolic pathways KEY CONCEPTS • Prokaryotes regulate gene expression in response to environmental stimuli KEY CONCEPTS • Gene regulation in prokaryotes occurs primarily at the transcription level Learning Objective 6 • Discuss the structure of a typical eukaryotic gene and the DNA sequences involved in regulating that gene Eukaryotic Genes • Are not normally organized into operons • Regulation occurs at levels of • • • • Transcription mRNA processing Translation Modifications of protein product Transcription • Requires • Transcription initiation site • where transcription begins • Promoter • to which RNA polymerase binds • In multicellular eukaryotes • RNA polymerase binds to promoter (TATA box) Transcription TATA box UPE T T TATA A A A Transcription initiation site pre-mRNA (a) Eukaryotic promoter elements. Fig. 14-9a, p. 316 TATA box UPE T T TATA A A A Transcription initiation site pre-mRNA (b) A weak eukaryotic promoter. Fig. 14-9b, p. 316 UPE UPE UPE UPE TATA box T T TATA A A A Transcription initiation site pre-mRNA (c) A strong eukaryotic promoter. Fig. 14-9c, p. 316 Enhancer UPE UPE TATA box T T TATA A A A Transcription initiation site pre-mRNA (d) A strong eukaryotic promoter plus an enhancer. Fig. 14-9d, p. 316 Regulated Eukaryotic Gene • Promoter • • • RNA polymerase-binding site short DNA sequences (upstream promoter elements (UPEs) or proximal control elements) UPEs • number and types within promoter region determine efficiency of promoter Enhancers (1) • Located far away from promoter • • control some eukaryotic genes Help form active transcription initiation complex Enhancers (2) • Specific regulatory proteins • • bind to enhancer elements activate transcription by interacting with proteins bound to promoters Enhancers Enhancer Target proteins RNA polymerase TATA box DNA (a) Little or no transcription. Fig. 14-11a, p. 317 Enhancer DNA TATA box Activator (transcription factor) (b) High rate of transcription. Fig. 14-11b, p. 317 Learning Objective 7 • In what ways may eukaryotic DNA-binding proteins bind to DNA? Transcription Factors • DNA-binding protein regulators control eukaryotic genes • • some transcriptional activators some transcriptional repressors Transcription Factors • Each has DNA-binding domain • 3 types of regulatory proteins • • • Helix-turn-helix Zinc fingers Leucine zippers Helix-Turn-Helix • Inserts one helix into DNA Turn α -helix DNA (a) Helix-turn-helix. Fig. 14-10a, p. 317 Zinc Fingers • Loops of amino acids • • held together by zinc ions each loop has α-helix that fits into DNA COO– Finger 2 Finger 3 Zinc ion Finger 1 NH3+ DNA (b) Zinc fingers. Fig. 14-10b, p. 317 Leucine Zipper Proteins • Associate as dimers that insert into DNA Leucine zipper region DNA (c) Leucine zipper. Fig. 14-10c, p. 317 Learning Objective 8 • How may a change in chromosome structure affect the activity of a gene? Gene Activity (1) • Changes in chromosome structure • • inactivates genes Heterochromatin • • densely packed regions of chromosomes contain inactive genes Gene Activity (2) • Active genes • • associated with loosely packed chromatin structure (euchromatin) Cells change chromatin structure • • from heterochromatin to euchromatin by chemically modifying histones (proteins associated with DNA to form nucleosomes) Chromatin Structure Heterochromatin: genes silent Chromatin decondensation Nucleosome Histones DNA Transcribed region Euchromatin: genes active Fig. 14-7, p. 314 Gene Activity (3) • Histone tail • • string of amino acids that extends from the DNA-wrapped nucleosome Methyl groups, acetyl groups, sugars, and proteins • • may chemically attach to the histone tail may expose or hide genes (turn on or off) Gene Activity (4) • Epigenetic inheritance • • • changes how a gene is expressed important mechanism of gene regulation DNA methylation • • • perpetuates gene inactivation patterns repeat in successive cell generations mechanism for epigenetic inheritance Gene Amplification • Some genes • • • products are required in large amounts have multiple copies in the chromosome Gene amplification • some cells selectively amplify genes by DNA replication Gene Amplification Drosophila chorion gene Gene amplification by repeated DNA replication of chorion gene region Chorion gene in ovarian cell Fig. 14-8, p. 315 Learning Objective 9 • How may a gene in a multicellular organism produce different products in different types of cells? Differential mRNA Processing • Single gene produces different forms of protein in different tissues • • depending on how pre-mRNA is spliced Gene contains a segment that can be either intron or exon • • as intron, sequence is removed as exon, sequence is retained Differential mRNA Processing Potential splice sites Exon Intron Exon or intron Exon pre-mRNA Differential mRNA processing Exon Exon Exon Functional mRNA in tissue A Exon Exon Functional mRNA in tissue B Fig. 14-12, p. 318 Learning Objective 10 • What types of regulatory controls operate in eukaryotes after mature mRNA is formed? mRNA Stability • Certain regulatory mechanisms increase RNA stability • • allowing more protein synthesis before mRNA degradation Sometimes under hormonal control Posttranslational Control (1) • In eukaryotic gene expression • • • feedback inhibition modification of protein structure Protein function change • • by kinases adding phosphate groups by phosphatases removing phosphates Protein Degradation (1) • Proteins targeted for destruction • • covalently bonded to ubiquitin Protein tagged by ubiquitin • degraded in a proteasome Protein Degradation (2) • Proteasome • • • large macromolecular structure recognizes ubiquitin tags Proteases • • • protein-degrading enzymes associated with proteasomes degrade protein into peptide fragments Protein Degradation Target protein Ubiquitin 1 Ubiquitin molecules attach to protein targeted for degradation. 2 Protein enters proteasome. Ubiquitinylated protein Proteasome 3 Ubiquitins are released and available for reuse. Protein is degraded into peptide fragments. Peptide fragments Fig. 14-13, p. 318 Target protein Ubiquitin 1 Ubiquitin molecules attach to protein targeted for degradation. Ubiquitinylated protein 2 Protein enters proteasome. Proteasome 3 Ubiquitins are released and available for reuse. Protein is degraded into peptide fragments. Peptide fragments Stepped Art Fig. 14-13, p. 318 KEY CONCEPTS • Gene regulation in eukaryotes occurs at the levels of transcription, posttranscription, translation, and posttranslation Animation: Controls of Eukaryotic Gene Expression CLICK TO PLAY