Topic 5 Regulation gene expression_CConstantinou_2024_Final.pdf

Topic 5. Regulation of Gene Expression Regulation of chromatin structure, regulation of transcription, post-transcriptional regulation, post-translational regulation, noncoding RNAs, operons Prof Constantina Constantinou [email protected] Monday 26th - Tuesday 27th of February 2024 Copyrigh...

Topic 5. Regulation of Gene Expression Regulation of chromatin structure, regulation of transcription, post-transcriptional regulation, post-translational regulation, noncoding RNAs, operons Prof Constantina Constantinou [email protected] Monday 26th - Tuesday 27th of February 2024 Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings Learning objectives (LOBs) 1. Describe the mechanisms involved in the regulation of gene expression in prokaryotes 2. Describe the mechanisms involved in the regulation of gene expression in eukaryotes 3. Describe the role of non-coding RNAs in the regulation of gene expression 4. Describe the role of abnormal gene expression in carcinogenesis. Reading: Chapter 18 Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings Lecture 1 Overview: Conducting the Genetic Orchestra Prokaryotes and eukaryotes change their gene expression in response to environmental changes In multicellular eukaryotes, gene expression regulates development and is responsible for differences between different cell types (cell specialization) RNA molecules play many roles in regulating gene expression in eukaryotes Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings 1. Regulation of gene expression in prokaryotes Bacteria respond to environmental change by regulating transcription Natural selection has favored bacteria to produce only the products that they need Bacteria can regulate metabolic pathways by: 1. Regulation of enzyme activity by feedback inhibition: controlled by allosteric regulation 2. Regulation of enzyme production by gene expression regulation: controlled by the operons Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings Example of gene expression regulation: Regulation of amino acid tryptophan in E.coli Precursor Feedback inhibition trpE gene Rapid response Enzyme 1 trpD gene Regulation of gene expression Enzyme 2 trpC gene Longer term response trpB gene Enzyme 3 trpA gene Tryptophan (a) Regulation of enzyme activity (b) Regulation of enzyme production Fig. 18-2 Operons: The Basic Concept Operon: a prokaryotic DNA segment that includes: ▪ The operator ▪ The promoter ▪ A group of functionally related genes Operator: ▪ A segment of DNA which works as a regulatory (on-off) switch that controls a cluster of functionally related genes ▪ Consists of a specific sequence within a promoter of these genes Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings trp operon trp operon Promoter Genes of operon trpE Operator Start codon mRNA 5 RNA polymerase E trpD trpC trpB trpA B A Stop codon D C Polypeptide subunits that make up enzymes for tryptophan synthesis Operon: a prokaryotic DNA segment that includes: ▪ The operator (regulator on/off switch) ▪ The promoter ▪ A group of functionally related genes Fig. 18-3a trp operon trp operon Promoter Genes of operon trpE Operator Start codon mRNA 5 RNA polymerase E trpD trpC trpB trpA B A Stop codon D C Polypeptide subunits that make up enzymes for tryptophan synthesis A simple switch off signal can switch off production of all enzymes involved in the synthesis of tryptophan Fig. 18-3a Operons: The Basic Concept Repressor: ▪ A protein that switches off the operon ▪ Prevents gene transcription by binding to the operator and blocking RNA polymerase binding ▪ Produced by a separate regulatory gene ▪ Can be in an active or inactive form, depending on the presence of other molecules Co-repressor: ▪ A molecule that cooperates with a repressor protein to switch an operon off (operon inactivation) Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings Negative gene regulation: Repressible and Inducible Operons Negative gene regulation: operons are switched off by the active form of the Repressor ▪ Repressible operons o Usually active o Usually regulate gene expression of enzymes involved in anabolic pathways o Their synthesis is repressed by high levels of the end product (corepressor) which activates the repressor o Example: the trp operon ▪ Inducible operons: o Usually inactive o Usually regulate gene expression of enzymes involved in catabolic pathways o Their synthesis is induced by a chemical signal (inducer) which inactivates the repressor o Example: the lac operon Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings Repressible operons: The trp (tryptophan) operon E. coli can synthesize the amino acid tryptophan Trp operon: contains genes of enzymes involved in tryptophan synthesis Repressible operon: Transcription is normally on but can be inhibited (repressed) when a small molecule (tryptophan) binds allosterically to a regulatory protein Tryptophan absence: the trp operon is activated ▪ The repressor is inactive => cannot bind the operator ▪ The genes for enzymes required for tryptophan synthesis are transcribed ▪ Tryptophan production starts Tryptophan presence: the trp operon is inactivated ▪ trp is a corepressor => binds to the trp repressor protein ▪ the repressor is activated => binds the operator ▪ trp operon is inactivated ▪ Tryptophan production stops Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings Repressible operons: The trp (tryptophan) operon The repressor is active only in the presence of its corepressor tryptophan Thus the trp operon is turned off (repressed) if tryptophan levels are high Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings trp operon activation in the absence of tryptophan trp operon Promoter Promoter Genes of operon DNA trpR Regulatory gene mRNA 5 Protein trpE 3 Operator Start codon mRNA 5 RNA polymerase Inactive repressor E trpD trpC trpB trpA B A Stop codon D C Polypeptide subunits that make up enzymes for tryptophan synthesis (a) Tryptophan absent: repressor inactive => repressor does not bind to operator => operon on => RNA polymerase transcribes genes => production of enzymes involved in tryptophan synthesis => tryptophan synthesis Fig. 18-3a trp operon inactivation in the presence of tryptophan operator DNA No RNA made mRNA Protein Active repressor Tryptophan (corepressor) (b) Tryptophan present: tryptophan binds to repressor => repressor active => operon off=> RNA polymerase cannot transcribe genes => production of enzymes involved in tryptophan synthesis is inhibited => no production of tryptophan Fig. 18-3b-2 Inducible Operons: the lac operon Lac operon: an inducible operon which contains genes that code for enzymes used in lactose metabolism (hydrolysis) Inducible operon: Transcription is normally off but can be activated (induced) when a small molecule (lactose) binds allosterically to a regulatory protein Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings Inducible Operons: the lac operon Lactose absence: lac operon is inactivated The lac repressor is active by itself => binds to the operator lac operon is inactivated => lactose hydrolysis stops Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings lac operon inactivation in the absence of lactose (a) Lactose absent => repressor active => repressor binds to operator => operon off => RNA polymerase cannot transcribe genes => inhibition of transcription of genes involved in lactose metabolism => no lactose hydrolysis Fig. 18-4 Inducible Operons: the lac operon Lactose presence: lac operon is activated Inducer: a molecule that binds and inactivates the repressor Allolactose is a disaccharide similar to lactose (isomer). It consists of the monosaccharides D-galactose and D-glucose linked through a β1-6 glycosidic linkage instead of the β1-4 linkage of lactose Allolactose = inducer of lac operon=> inactivates repressor →The inducer turns the lac operon on The genes for enzymes involved in lactose hydrolysis are transcribed → Lactose hydrolysis starts i.e. lactose is broken down to glucose and galactose Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings lac operon activation in the presence of lactose Lactose= glucose and galactose (b) Allolactose present=>allolactose binds to the repressor and inactivates it => repressor inactive => operon on => transcription of genes involved in lactose metabolism => breakdown of lactose to glucose and galactose Positive Gene Regulation Positive gene regulation: operons are switched on by the active form of the activator Activator of transcription: a stimulatory protein ▪ Example: Catabolite Activator Protein (CAP) in E. coli which enhances transcription of the lac operon Lac operon: ▪ Active in the presence of lactose (negative gene regulation) ▪ Glucose absence further stimulates its transcription (positive gene regulation) Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings Positive Gene Regulation The enzymes for glucose breakdown in glycolysis are continually present When glucose and lactose are both present, E.coli will preferentially use glucose rather than lactose => in glucose and lactose presence => low quantities of the enzymes which are needed for lactose breakdown (normal low transcription) Only when lactose is present and glucose is in short supply does E.coli use lactose as an energy source and it is only then that it synthesizes in sufficient quantities the enzymes which are needed for lactose breakdown (enhanced transcription) Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings Positive Gene Regulation Low glucose levels: ▪ Increase in levels of cAMP ▪ CAP is activated by binding to cAMP ▪ Activated CAP attaches to the promoter of the lac operon, increases the affinity of RNA polymerase and accelerates transcription of the lac operon (genes producing proteins involved in hydrolysis of lactose to glucose and galactose) High glucose levels ▪ Decrease in levels of cAMP ▪ CAP detaches from the lac operon ▪ Decreased affinity of RNA polymerase and decreased (low) transcription of the lac operon (genes producing proteins involved in hydrolysis of lactose to glucose and galactose) Positive control of the lac operon by CAP Promoter DNA lacI lacZ CAP-binding site cAMP Active CAP Inactive CAP Allolactose RNA polymerase binds and transcribes Operator Inactive lac repressor (a) Lactose present, glucose scarce => cAMP level high=> activation of CAP => increased affinity of RNA polymerase => abundant lac mRNA synthesized Positive control of the lac operon by CAP Promoter DNA lacI lacZ Operator CAP-binding site RNA polymerase less likely to bind Inactive CAP Inactive lac repressor Allolactose (b) Lactose present, glucose present=> cAMP levels low=> inactivation of CAP => decreased affinity of RNA polymerase => little lac mRNA synthesized (normal rate) Figure 18 Prokaryotic gene expression regulation Summary: Operons Negative gene regulation: operons are switched off by the active form of the repressor ▪ Repressible Operons: e.g. trp operon regulation by tryptophan: o Tryptophan absent => repressor inactive => operon on o Tryptophan present => repressor active => operon off ▪ Inducible Operons: e.g. lac operon regulation by lactose: o Lactose absent => repressor active => operon off o Lactose present => repressor inactive => operon on Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings Prokaryotic gene expression regulation Summary: Operons Positive gene regulation: operons are switched on by the active form of the activator. E.g. lac operon regulation by glucose o Glucose absent/low + lactose present => cAMP high levels => CAP active => increased affinity of RNA polymerase => transcription accelerated (operon overactivated) o Glucose present + lactose present => cAMP low levels => CAP inactive => decreased affinity of RNA polymerase => normal low rate of transcription (operon on- normal low activation) Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings Prokaryotic gene expression regulation summary: Trp operon (Negative gene regulation) Trp operon: Tryptophan present Tryptophan absent Repressor active Repressor inactive operon off Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings operon on Prokaryotic gene expression regulation summary: Lac operon (Negative gene regulation) Lac operon regulation by lactose: Lactose absent Lactose present Repressor active Repressor inactive operon off Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings operon on Prokaryotic gene expression regulation summary: Lac operon (Positive gene regulation) Lac operon regulation by glucose: Glucose absent/low (lactose present) High cAMP levels Low cAMP levels CAP active operon over-activated (transcription accelerated) Glucose present (lactose present) CAP inactive operon on (normal low rate of transcription) Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings 2. Regulation of gene expression in eukaryotes Eukaryotic gene expression can be regulated at any stage Gene expression in eukaryotes: ▪ Regulates development ▪ Is responsible for cell specialization (differentiation) Differential gene expression: the expression of different genes by cells with the same genome ▪ Different cell types produced Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings Differential Gene Expression Almost all the cells in an organism are genetically identical A typical human cell only expresses 20% of its genes at any given time Differences between cell types result from differential gene expression Only 1.5% of the DNA codes for proteins The rest of the DNA codes for RNA products (e.g. rRNAs and tRNAs) or is not transcribed at all Abnormalities in gene expression can lead to diseases (e.g. cancer) Gene expression is regulated at many stages Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings Signal Eukaryotic gene expression can be regulated at any stage NUCLEUS Chromatin Chromatin modification DNA Regulation of chromatin structure ▪ Histone acetylation and DNA methylation Gene available for transcription Gene Transcription RNA Exon Primary transcript Intron RNA processing Tail Regulation of transcription initiation Cap mRNA in nucleus Transport to cytoplasm Post-transcriptional regulation ▪ RNA processing, mRNA degradation, initiation of translation, protein processing and degradation CYTOPLASM mRNA in cytoplasm Degradation of mRNA Translation Polypeptide Protein processing Active protein Degradation of protein Transport to cellular destination Cellular function Fig. 18-6 1. Regulation of Chromatin structure 1. Regulation of Chromatin structure The Human DNA is 6.3 Gigabase (109) pairs (Gbp) long, 205 cm long and weighs 6.4 picograms (10-12) (pg) per cell The DNA of eukaryotic cells is packaged with the proteins histones in an elaborate complex known as chromatin Euchromatin: ▪ The active form of chromatin (loosely packed) ▪ Gene expression activated Heterochromatin: ▪ The inactive form of chromatin (highly packed) ▪ Gene expression inactivated 1. Regulation of Chromatin structure Chromatin modifications: Chemical modifications to histones and DNA of chromatin influence chromatin structure => gene expression Epigenetic inheritance: the inheritance of traits transmitted by mechanisms independent of nucleotide sequence changes (e.g. chromatin modifications) Chromatin-modifying enzymes: control gene expression by increasing or decreasing the ability of the transcription machinery to bind to the DNA (e.g. Histone acetyltransferases , Histone deacetylases -see Topic 3) Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings 1. Regulation of Chromatin structure Histone Modifications DNA methylation Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings Nucleosome ▪ A nucleosome is the basic structural unit of DNA packaging in eukaryotes It consists of a segment of DNA (a little less than two turns of DNA wrapped) around a set of eight proteins called histones, which are known as a histone octamer (two copies each of the histone proteins H2A, H2B, H3 and H4). Histone Modifications (Acetylation) Histone acetylation ▪ Implemented by Histone acetylation enzymes which promote the initiation of transcription by remodelling the chromatin structure and by recruiting the transcription machinery ▪ The N- terminus of each histone molecule in a nucleosome protrudes outward from the nucleosome ▪ Acetyl groups (-COCH3) are attached to (+) charged lysines in histone tails ▪ When the lysines are acetylated, their positive charges are neutralized and the histone tails do not bind to neighboring nucleosomes ▪ Chromatin has a looser structure ▪ Activation of transcription Histone deacetylation ▪ Implemented by histone deacetylases (HDACs) ▪ Removal of Acetyl groups (-COCH3) restores the histone (+) charge, which causes increased binding to neighbouring nucleosomes and hence the inactive form of chromatin ▪ Inactivation of transcription Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings Acetylation of histone tails Histone tails DNA double helix Amino acids available for chemical modification (a) Histone tails protrude outward from a Nucleosome.The aminoacids in the N terminal tails are available for chemical modification. Acetylation of histone tails ▪ Acetyl groups (-COCH3) are attached to (+) charged lysines in histone tails ▪ When the lysines are acetylated, their positive charges are neutralized and the histone tails do not bind to neighboring nucleosomes ▪ Chromatin has a looser structure ▪ Activation of transcription Unacetylated histones histones Acetylated (b) Acetylation of histone tails promotes loose chromatin structure that permits transcription Histone Modifications (Methylation and Phosphorylation) Histone methylation ▪ Addition of methyl groups (-CH3) (nonpolar group/ neither negative or positive) in an amino acid (lysine or arginine) in the histone ▪ Chromatin condensation ▪ Gene expression inactivation ▪ *Even though histone methylation is in general associated with transcriptional repression, methylation of some lysine and arginine residues of histones results in transcriptional activation. Phosphorylation ▪ Addition of a phosphate group to an amino acid which is next to a methylated amino acid ▪ Decondenses chromatin ▪ Activation of transcription Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings DNA Methylation DNA methylation: ▪ The addition of methyl groups (-CH3) to certain bases in DNA (usually cytosine) leads to reduced transcription ▪ Can cause long-term inactivation of genes involved in cellular differentiation ▪ Comparison of the same genes in different tissues shows that the genes are usually more heavily methylated in cells in which they are not expressed Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings DNA Methylation Genomic imprinting: ▪ Genomic imprinting occurs during the formation of the gametes and results in the silencing (inactivation) of either the paternal or maternal alleles of certain genes by methylation (addition of methyl groups to cytosine nucleotides). ▪ Because the genes are imprinted differently in sperm and eggs, a zygote expresses only one allele of an imprinted gene (either allele inherited from the mother or allele inherited from the father) ▪ Either the maternal or the paternal allele of the gene of a given imprinted gene is expressed in every cell of the organism ▪ Only affects a small fraction of mammalian genes (1%) Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings DNA Methylation Genomic imprinting: ▪ IGF2 is a growth factor which is essential for embryonic/ fetal development. o Imprinted (inactivated) in humans--> maternal allele normally silenced by methylation o Abnormal activation of maternal IgF2 allele during egg formation/early development--> Beckwith-Wiedemann Syndrome (BWS)→ increased risk of cancer (1:15000 births) Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings Genomic Imprinting Insulin like growth factor 2 (IgF2) is required for prenatal development and only the paternal allele is expressed Paternal chromosome Normal Igf2 allele is expressed Maternal chromosome Normal Igf2 allele is not expressed Wild-type mouse (normal size) (a) Homozygote A mouse homozygous for the wild type igF2 allele is normal sized. Only the paternal allele of the gene is expressed. Copyright © 2008 Pearson Educat ion Inc., publishing as Pearson BenjaminCummings Genomic Imprinting Mutant Igf2 allele inherited from mother Mutant Igf2 allele inherited from father Normal size mouse (wild type) Normal paternal allele expressed Normal Igf2 allele is expressed Mutant maternal allele silenced Mutant Igf2 allele is not expressed Dwarf mouse (mutant) Mutant Igf2 allele is expressed Dwarf mouse due to silencing of normal maternal allele Mutant paternal allele expressed Normal maternal Normal Igf2 allele allele silenced is not expressed Heterozygotes Copyright © 2008 Pearson Educat ion Inc., publishing as Pearson(b) BenjaminCummings Matings between wild-type mice and those homozygous for the recessive mutant IgF2 allele produce heterozygous offspring. The mutant phenotype is seen only when the father contributed the mutant allele because the maternal allele is not expressed. Lecture 2 2. Regulation of Transcription Initiation Organization of a Typical Eukaryotic Gene Promoter: ▪ A DNA sequence where RNA polymerase II and transcription factors bind ▪ Present in each eukaryotic gene ▪ Located upstream of the gene ▪ Includes TATA box Control elements: ▪ Segments of non-coding DNA that regulate transcription by binding to transcription factors o Proximal control elements: located close to the promoter o Distal control elements (grouped together as enhancers): located far away from a gene or even within an intron Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings Organization of a Typical Eukaryotic Gene Transcription factors: Proteins which help RNA polymerase II to initiate transcription Interact with specific control elements => regulate transcription of particular genes Control elements + transcription factors => regulation of gene expression in different cell types Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings Organization of a Typical Eukaryotic Gene Enhancer (distal control elements) Poly-A signal sequence Termination region Proximal control elements Exon Intron Exon Intron Exon DNA Upstream Downstream Promoter Primary RNA 5 transcript Transcription Exon Intron Exon Intron Exon RNA processing Cleaved 3end of primary transcript Poly-A signal Intron RNA Coding segment mRNA 3 5Cap 5UTR Start codon Stop codon 3UTR Poly-A tail The Roles of Transcription Factors Transcription factors: ▪ Help RNA polymerase II to initiate transcription ▪ Essential for the transcription of all protein-coding genes 2 types of specific transcription factors: ▪ Activators: transcription factors that bind to an enhancer and stimulate specific gene transcription ▪ Repressors: transcription factors that inhibit transcription and expression of a particular gene Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings Enhancers and Specific Transcription Factors (TFs) 1. Activators bind to an enhancer (control element) to stimulate specific gene transcription 2. Bound activators cause recruitment of mediator proteins 3. Recruitment of general TFs which bind to TATA box within the promoter 4. Recruitment of RNA polymerase II which binds to the promoter 5. Activation of gene transcription Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings Promoter Activators DNA Enhancer 1. Activator proteins bind to distal control elements (enhancer) in the DNA. This enhancer has three binding sites. Distal control element TATA box General transcription factors DNA-bending protein Group of mediator proteins 2. DNA bending protein brings the bound activators closer to the promoter. Recruitment of mediator proteins, General transcription factors and RNA polymerase II nearby. 3. The activators bind to mediator proteins and general transcription factors, helping them form an active transcription initiation complex on the promoter Gene RNA polymerase II RNA polymerase II Transcription initiation complex RNA synthesis Fig. 18-9-3 Activator structure Activation domain DNA-binding domain DNA Activators have 2 domains: a DNA-binding domain and an activation domain (activates transcription) Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings Figure 18.9 Types of Transcription Factors Specific transcription factors (Activators): ▪ Unique for each gene (only common for the functionally related genes that need to be coexpressed) ▪ Once these TFs bind to the control elements (e.g. enhancers), they cause recruitment and binding of the general transcription factors to the TATA box General transcription factors: ▪ Common for all the genes ▪ Bind to the TATA box in order to induce RNA polymerase II binding to the promoter Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings Co-expression of genes in prokaryotes vs eukaryotes Prokaryotes: Functionally related genes of a prokaryotic operon are regulated by the same promoter Production of polycistronic mRNA molecule (encodes more than one polypeptide)→ co-expressed Eukaryotes: Each eukaryotic gene has its own promoter and control elements Production of monocistronic mRNA (encodes for only one polypeptide) Functionally related genes have the same control elements and activators even if located on different chromosomes Activators recognise specific control elements and promote simultaneous transcription of genes→ co-expressed Coordinately Controlled Genes in Eukaryotes Combinatorial Control of Gene Activation: ▪ A combination of control elements allow the activation of transcription only when the appropriate activator proteins are present Example: Both liver cells and lens cells have the genes for making the proteins albumin and crystalline but only liver cells make albumin and only lens cells make crystalline Tissue specific gene expression in eukaryotes Enhancer Control elements Promoter DIFFERENTIAL GENE EXPRESSION Albumin gene Crystallin gene LENS CELL NUCLEUS LIVER CELL NUCLEUS All the activators required for high expression of albumin are only present in liver cells Available activators Available activators Albumin gene not expressed Albumin gene expressed Crystallin gene not expressed (a) Liver cell Crystallin gene expressed (b) Lens cell All the activators required for high expression of crystallin are only present in lens cells 3. Mechanisms of Post-Transcriptional Regulation Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings 3. Mechanisms of Post-Transcriptional Regulation Transcription alone does not account for gene expression Post-transcriptional regulation: ▪ Regulatory mechanisms that operate at various stages after transcription ▪ Provide rapid regulation of gene expression in response to environmental changes Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings 1. RNA Processing 1. RNA Processing Alternative RNA splicing: Production of different mRNAmolecules from the same primary RNA transcript depending on which RNA segments are treated as exons and which as introns Production of different proteins from the same primary RNA transcript β-thalassaemia: some types due to abnormal splicing of the βglobin gene PremRNA Exon 4 treated as intron=>removed Exon 3 treated as intron=>removed Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings Alternative Splicing Exons DNA Troponin T gene Primary RNA transcript RNA splicing Exon 4 treated as intron=>removed Exon 3 treated as intron=>removed mRNA Two different muscle proteins are produced 2. mRNA degradation 2. mRNA degradation The life span of mRNA molecules in the cytoplasm is a key to determining protein synthesis Eukaryotic mRNA is more long lived than prokaryotic mRNA The mRNA life span is determined in part by sequences in the 3’-untranslated region (3'-UTR) Nucleases and non-coding RNAs induce mRNA degradation => Inhibition of gene expression Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings 3. Initiation of Translation 3. Initiation of Translation Translation of all mRNAs in a cell may be regulated simultaneously (e.g. following egg fertilization) The initiation of translation of selected mRNAs can be blocked by regulatory proteins that bind to sequences or structures within the unstranslated 5’UTR region of the mRNA preventing the attachment of the ribosomes Non-coding RNAs can also inhibit the initiation of translation Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings 4. Protein Processing and Degradation 4. Protein Processing and Degradation Post-translational modifications: polypeptide processing in order to produce functional proteins ▪ Polypeptide cleavage: some polypeptides are activated by enzymes that cleave them (e.g. insulin) -in RER ▪ Protein folding (tertiary structure): e.g. disulphide bond formation- in RER ▪ Subunit assembly (quaternary structure): Some polypeptides come together to form the subunits of a functional protein (e.g. hemoglobin) -in RER ▪ Chemical modifications: addition of chemical groups to proteins → formation of glycoproteins, lipoproteins - some in RER but mostly in Golgi apparatus Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings Protein degradation: Proteasome Protein degradation: ▪ To mark a particular protein for destruction, the cell attaches the small protein ubiquitin to the protein e.g. cyclins in the cell cycle ▪ Non-functional (misfolded) proteins are also attached to ubiquitin (ubiquitination) ▪ Ubiquitinated proteins are targeted to the proteasome for degradation Proteasomes: giant protein complexes that bind to protein molecules and degrade them (size= 26S) Long lived proteins are degraded in the lysosome Protein degradation: Proteasome Ubiquitin Proteasome Protein to be degraded Ubiquitinated protein Proteasome and ubiquitin to be recycled Protein entering a proteasome Protein fragments (peptides) Fig. 18-12. Degradation of a protein by a proteasome. 3. Control of gene expression by non-coding RNAS Non-coding RNAs play multiple roles in controlling gene expression Only 1.5% of the DNA codes for proteins (Coding DNA) The rest of the DNA codes for RNA products (e.g. rRNAs and tRNAs) or is not transcribed at all (Non-coding DNA) Non-coding DNA: transcribed but not translated ▪ Part of it has the genes for rRNA and tRNA ▪ A significant amount of the genome transcribed into noncoding RNAs (ncRNAs) Function of noncoding RNAs: regulate mRNA translation and chromatin configuration => regulate gene expression Effects of miRNAs and siRNAs on mRNA MicroRNAs (miRNAs): ▪ Small single-stranded RNA molecules (20-25bp) that can bind to mRNA ▪ They can degrade mRNA or block its translation Small interfering RNAs (siRNAs): ▪ Small double-stranded RNA molecules (20-25 bp) that bind to the mRNA ▪ Cause RNA interference (RNAi): inhibition of gene expression by RNA molecules siRNAs and miRNAs are similar but form from different RNA precursors Generation and function of miRNAs Hairpin miRNA Hydrogen bond Dicer miRNA 5 3 (a) Primary miRNA transcript Less likely miRNAprotein complex Most likely miRNA- incomplete base pairing with many mRNAs => mostly block translation siRNA- complete base pairing with specific mRNAs=> mRNA degradation mRNA degraded Translation blocked Fig. 18-13 Chromatin Remodeling and Silencing of Transcription by Small RNAs Functions of siRNAs: ▪ Chromatin modification: induce heterochromatin formation => can block large regions of the chromosome by inhibition of transcription and gene expression ▪ Transcription regulation: inhibit transcription of specific genes Comparison of miRNA and siRNA ncRNA type Structure Function Base pairing with target mRNA miRNA Single stranded RNA molecule (20-25 nucleotides) Mostly inhibits translation Incomplete base pairing with many different mRNAs => can target various mRNAs and inhibit expression of various genes siRNA Double stranded RNA molecule (20-25 base pairs) Mostly causes mRNA degradation Complete base pairing with a specific mRNA => can inhibit expression of specific genes only (highly specific) Lecture 3 4. Genetic changes that affect the cell cycle and lead to cancer Cancer Cancer is an abnormal proliferation of cells in an uncontrolled manner Carcinogenesis is caused by abnormal function of the gene regulation systems Cancer can be caused by mutations to genes that regulate cell division (cell cycle) Tumour viruses (oncogenic viruses) can cause cancer in animals including humans by insertion of their genome into the cellular genome Viruses associated with human cancer DNA viruses Epstein-Barr virus Burkitt’s lymphoma Nasopharyngeal carcinoma Human Papilloma viruses Cervical carcinoma, warts Hepatitis B virus Hepatocellular carcinoma RNA retroviruses HTLV-I Adult T-cell leukaemia lymphoma Oncogenic Retroviruses Acutely transforming: -e.g. Avian Erythroblastosis Virus (v-erb-b2), Avian Myelocytomatosis Virus (v-myc) -Cause tumours by transduction of the viraloncogene (v-Onc) The virus genome Viralgenes Viralpromoter contains an Viral Gag Pol v-Onc oncogene genome Cellular DNA Cellular DNA Gag Pol v-Onc The viral oncogene is constitutively active By inserting into the genome of the cell it causes uncontrolled proliferation of cells that leads to carcinogenesis Oncogenic Retroviruses Non-defective(slowly transforming): - e.g. Mouse Mammary TumourVirus(MMTV), Avian Leukosis Virus(ALV) - Cause tumours by insertional mutagenesis The virus genome does not containan oncogene promoter Viral genome Cellular DNA Gag Pol Env It insertsitself upstream of c-myc Viral promoter Gag Cellular DNA Pol Env c-myc = cellular proto-oncogene responsible for normal cell proliferation c-myc The strong viral promoter causes uncontrolled cell proliferation and therefore carcinogenesis Types of Genes Associated with Cancer Oncogenes: ▪ Genes found in viral or cellular genomes that trigger the molecular events that can lead to cancer →Oncogenes induce uncontrolled cell division and therefore promote cancer development Tumour suppressor genes: ▪ Genes whose protein product inhibits cell division → Tumour suppressor genes prevent uncontrolled cell division and therefore prevent cancer development Oncogenes and proto-oncogenes Oncogenes: cancer-causing genes Proto-oncogenes: the corresponding normal cellular genes that are responsible for normal cell growth and division (e.g. growth factors, receptors, intracellular molecules of signalling pathways) Conversion of a proto-oncogene to an oncogene can lead to abnormal stimulation of the cell cycle The Cell Cycle and Oncogenes Proto-oncogenes ON/ OFF Controlled cell division Mutations Oncogenes ON Uncontrolled cell division Cancer Oncogenes and proto-oncogenes Proto-oncogenes can be converted to oncogenes by: 1. Movement of DNA within the genome (translocation/ transduction): e.g. in Chronic Myelogenous Luekemia (CML) ▪ DNA may be inserted downstream of an active promoter o Transcription of the genes that it encodes may increase ▪ Inserted DNA may contain an active promoter (e.g. viral promoter) o Transcriptional activation of genes Oncogenes and proto-oncogenes Proto-oncogene DNA Translocation or transposition: Gene amplification: within a control element New promoter Normal growthstimulating protein in excess Point mutation: Oncogene Normal growth-stimulating protein in excess Normal growthstimulating protein in excess within the gene Oncogene Hyperactive or degradationresistant protein Fig. 18-20 Chromosomal translocations in Chronic Myeloid Leukaemia (CML) Translocation 9 22 Philadelphia chromosome Fusion between gene ABL (chromosome 9) and gene BCR (chromosome 22) produces an oncogene i.e. a protein with enhanced tyrosine kinase activity that leads to increased cell division and therefore tumorigenesis. 95% of cases of Chronic Myeloid Leukaemia (CML) have detectable Ph chromosome BCR-ABL1 BCR: encodes a protein that ABL encodes a protein acts as a guanine nucleotide tyrosine kinase whose activity exchange factor for Rho is tightly regulated (auto- GTPase proteins inhibition) BCR-ABL protein has constitutive (unregulated) protein tyrosine kinase activity which leads to uncontrolled cell division BCR-ABL1 Oncogenes and proto-oncogenes Proto-oncogenes can be converted to oncogenes by: 2. Amplification of a proto-oncogene: increased number of copies of the gene e.g. EGFR-2 amplification in HER-2 positive breast cancers ▪ Increased activation of the MAPK signaling pathway ▪ Increased cellular proliferation Oncogenes and proto-oncogenes Proto-oncogene DNA Translocation or transposition: Gene amplification: within a control element New promoter Normal growthstimulating protein in excess Point mutation: Oncogene Normal growth-stimulating protein in excess Normal growthstimulating protein in excess within the gene Oncogene Hyperactive or degradationresistant protein Fig. 18-20 Cell cycle–stimulating pathway: constitutively activated in cancer (The HER2 Oncogene) The HER2 Oncogene HER2/neu/ERBB2 gene encodes for part of the human epidermal growth factor receptor Growth factors bind EGFR or HER3 and alter conformation of receptors that become active Receptor dimerization is required for HER2 function The HER2 Oncogene HER-2 HER2 is amplified in ~20% of the invasive breast cancers and associated with aggressive disease and poor prognosis Trastuzumab (Herceptin) is a monoclonal antibody that targets HER2 (targeted therapy) Oncogenes and proto-oncogenes Proto-oncogenes can be converted to oncogenes by: 3. Mutations in the proto-oncogene or its control elements: gain-of-function mutations ▪ Increased gene expression of the proto-oncogene ▪ Constitutively active oncogene (e.g. Ras) Oncogenes and proto-oncogenes Proto-oncogene DNA Translocation or transposition: Gene amplification: within a control element New promoter Normal growthstimulating protein in excess Point mutation: Oncogene Normal growth-stimulating protein in excess Normal growthstimulating protein in excess within the gene Oncogene Hyperactive or degradationresistant protein Fig. 18-20 Cell cycle–stimulating pathway: constitutively activated in cancer (The Ras Oncogene) Mutations in the Ras proto-oncogene is common in human cancers Production of the Ras oncogene → hyperactive Ras protein → increased cell division → Cancer Cell cycle–stimulating pathway: constitutively activated in cancer (Ras Oncogene) 1 Growth factor MUTATION Ras 3 G protein GTP Ras GTP 2 Receptor Hyperactive Ras protein (product of oncogene) issues signals on its own MAPK proliferative signaling pathway 4 Protein kinases (phosphorylation cascade) NUCLEUS 5 Transcription factor (activator) DNA Gene expression Protein that stimulates the cell cycle Fig. 18-21a Oncogenes and proto-oncogenes Proto-oncogene DNA Translocation or transposition: Gene amplification: within a control element New promoter Normal growthstimulating protein in excess Point mutation: Oncogene Normal growth-stimulating protein in excess Normal growthstimulating protein in excess within the gene Oncogene Hyperactive or degradationresistant protein Fig. 18-20 Tumor-Suppressor Genes Tumor-suppressor genes: genes whose protein products inhibit cell division ▪ They prevent uncontrolled cell division →prevent cancer development ▪ Inactivation of tumour suppressor genes leads to cancer Tumour suppressor genes Cell division Tumor-Suppressor Genes Mechanism of inactivation of tumour suppressor genes: ▪ Loss-of function mutations: inactivating mutations within tumour-suppressor genes → inactive tumor suppressor genes→ inactive proteins → cancer development ▪ Insertional mutagenesis: insertion of viral genome into host cell DNA → inactive tumor suppressor genes→ inactive proteins → cancer development Roles of Tumor-suppressor proteins ▪ Inhibit cell-signaling pathways → > inhibit the cell cycle or induce apoptosis (e.g. Rb and p53 proteins) ▪ Repair damaged DNA (e.g. BRCA-1) ▪ Control cell adhesion The Cell Cycle and Tumor Suppressor genes Proto-oncogenes Mutations Cancer Oncogenes Inactive Tumour suppressor genes Controlled cell division Mutations Tumour suppressor genes Controlled cell division Healthy cells control their growth and will kill themselves if they become unhealthy... Normal cell Mutation(s) Activation of Tumour Suppressor Genes Cell containing mutation(s) Activation of Cell Cycle Checkpoints and inhibition of Cell Cycle Apoptosis Cell containing mutation(s) Cancer cells cannot control their growth and cannot kill themselves... Normal cell Mutations Cell containing excessive mutations Defective tumor suppressor genes Uncontrolled cell division / Defective pathway of apoptosis Cancer Integration of the genome of DNA cancer viruses (e.g. HPV) in the host DNA Viral DNA Host DNA DNA of the host + virus Parts of the genome of DNA cancer viruses can be integrated in the genome of the host even though this is a rare event (1/1000). As a consequence there may be e.g. an inactivation of a tumour suppressor gene. Interference with Normal Cell Signaling Pathways Cell cycle arrest (inhibition of the cell cycle) can be important in the case of damage to a cell’s DNA p53 is a very important tumor suppressor gene called ‘the gatekeeper of the genome’ p53 prevents a cell that has DNA damage from passing on mutations to its daughter cells by replicating Mutations in the p53 gene prevent cell cycle arrest →Uncontrolled cell proliferation of damaged cells → Cancer p53 function Cdk (catalytic subunit) Cyclin (regulatory subunit) DNA damage Cell cycle arrest (inhibition) p53 CDK inhibitor (CKI) inhibition on CDK kinase activity p53: major tumour suppressor protein activated by DNA damage => causes cell cycle arrest at G1 phase Regulation of the Cell Cycle by tumour suppressors 1 2 3 4 1 2 3 4 CDK inhibitor p53 Rb Growth factor (mitogen) signalling Expression of early response genes G1 cyclin-dependent kinase (CDK) activity Transcription of genes encoding proteins required for DNA synthesis p53 and Rb are tumour suppressors Cell cycle regulation upon DNA damage DNA damage detected at the checkpoints → cell cycle arrest/block G2/M Checkpoints This gives the opportunity to the cell to try to repair this damage If this is not possible, this will lead to apoptosis (programmed cell death) 1.Stop the cycle 2.Attempt DNA repair 3. Induce Apoptosis (Programmed Cell Death) G1/S Checkpoint Cell cycle–inhibiting pathway: inactivated in cancer (p53 tumor suppressor gene) 2 Protein kinases MUTATION 3 Active UV light form of p53 1 DNA damage in genome DNA Mutations in p53 lead to the production of an inactive protein that, cannot activate transcription→ no inhibition of cell cycle Protein that inhibits the cell cycle Fig. 18-21b Effects of mutations on oncogenes and tumor suppressor genes Activated oncogenes Inactive tumour suppressor genes EFFECTS OF MUTATIONS Protein overexpressed Cell cycle overstimulated Protein absent Increased cell division Cell cycle not inhibited Fig. 18-21c HER2 amplification and p53 inactivation in several cancer types HER-2 amplification: increased copy number of HER-2 genes => HER-2 overexpression 12% of breast cancers have amplified HER2 genes resulting in over expression of the receptor 23% of breast cancers have mutated p53 genes http://cancer.sanger.ac.uk/cosmic/gene/analysis?ln=ERBB2#ts p53 inactivation: a variety of mutations abolish p53 activity HER-2= EGFR-2 http://cancer.sanger.ac.uk/cosmic/gene/analysis?ln=TP53#ts 1 The Multistep Model of Cancer Development Multiple mutations are generally needed for full-fledged cancer Cancer incidence increases with age At the DNA level, a cancerous cell is usually characterized by at least one active oncogene and the mutation of several tumor-suppressor genes Incidence of cancer Multiple Hit Hypothesis Age Accumulation of somatic mutations in a series of genes (oncogenes, tumour suppressor genes) within a single cell, followed by selection for neoplastic (cancer) phenotype The relationship between age and cancer The Multistep Model of Cancer Development Colon Colon wall Normal colon epithelial cells 1 Loss of tumorsuppressor gene APC (or other) 4 Loss of tumor-suppressor gene p53 2 Activation of ras oncogene Small benign growth (polyp) 3 Loss of tumor-suppressor gene DCC 5 Additional mutations Larger benign growth (adenoma) Malignant tumor (carcinoma) Fig. 18-22 Inherited Predisposition and Other Factors Contributing to Cancer Individuals can inherit oncogenes or mutant alleles of tumoursuppressor genes Inherited mutations in the tumour-suppressor gene adenomatous polyposis coli (APC) are common in individuals with colorectal cancer Mutations in the BRCA1 or BRCA2 genes are found in at least half of inherited breast cancers In 1990 Mary-Claire King demonstrated that mutations in gene BRCA1 are associated with increased susceptibility in cancer. Oncogenes and tumour suppressor gene summary Oncogenes HER-2 (growth factor receptor) Ras (MAPK pathway protein) Tumour suppressor genes p53 (DNA repair, G1 arrest) Rb (G1 arrest) BRCA-1 and BRCA-2 (ds DNA repair) Summary Prokaryotic gene expression regulation: operons Eukaryotic gene expression regulation: ▪ Regulation of chromatin structure ▪ Histone acetylation, DNA methylation ▪ Regulation of transcriptional initiation ▪ Post-transcriptional regulation ▪ RNA processing, mRNA degradation, initiation of translation, protein processing and degradation Regulation of gene expression by noncoding RNAs Genetic changes that affect the cell cycle and lead to cancer ▪ Oncogenes and Tumour suppressor genes Copyright © 2008 Pearson Education Inc., publishing as Pearson BenjaminCummings