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Chapter 18: Gene expression
- 1. Chapter 18 Regulation ofGene Expression • Prokaryotes and eukaryotes alter gene expression in response to their changing environment • In multicellular eukaryotes, gene expression regulates development and is responsible for differences in cell types • RNA molecules play many roles in regulating gene expression in eukaryotes
- 2. Figure 18.2 Precursor Feedback inhibition Tryptophan (b) Regulationof enzyme production—saves energy by avoiding making proteins not in use (a) Regulation of enzyme activity Regulation of gene expression trpE trpD trpC trpB trpA Enzyme 1 Enzyme 2 Enzyme 3
- 3. Prokaryotes use Operons:The Basic Concept • A cluster of functionally related genes can be under coordinated control by a single on-off “switch” • The regulatory “switch” is a segment of DNA called an operator usually positioned within the promoter • An operon is the entire stretch of DNA that includes the operator, the promoter, and the genes that they control • The operon can be switched off by a protein repressor (some repressors need co-repressors to be active) • The repressor prevents gene transcription by binding to the operator and blocking RNA polymerase • The repressor is the product of a separate regulatory gene
- 4. Biosynthetic Operons &Catabolic Operons • BiosyntheticTryptophan Biosynthesis – Off when lots of tryptophan (product) around – Tryptophan molecules act as co-repressor (activates the repressor protein to turn off the promoter i.e. block RNA Polymerase) • CatabolicLactose Utilization – On when lactose (substrate) is present – Lactose molecule inactivates the repressor protein to RNA Polymerase can transcribe
- 5. Figure 18.3 Promoter DNA trpR Regulatory gene RNA polymerase mRNA 5 3 Protein Inactive repressor mRNA5 (a) Tryptophan absent, repressor inactive, operon on DNA mRNA Protein Active repressor No RNA made Promoter trp operon Genes of operon trpE trpD trpC trpB trpA Operator Start codon Stop codon trpR trpE Tryptophan (corepressor) (b) Tryptophan present, repressor active, operon off 3 5 Polypeptide subunits that make up enzymes for tryptophan synthesis E D C B A
- 6. Repressible and InducibleOperons: Two Types of Negative Gene Regulation (= operator+repressor) • A repressible operon is one that is usually on; binding of a repressor to the operator shuts off transcription • The trp operon is a repressible operon • An inducible operon is one that is usually off; a molecule called an inducer inactivates the repressor and turns on transcription
- 7. • The lacoperon is an inducible operon and contains genes that code for enzymes used in the hydrolysis and metabolism of lactose • By itself, the lac repressor is active and switches the lac operon off • A molecule called an inducer inactivates the repressor to turn the lac operon on
- 8. Figure 18.4 Promoter DNA Regulatory gene mRNA 5′ 3′ Operator RNA polymerase Active repressor No RNA made IacZ lacZ lacYlacA (a) Lactose absent, repressor active, operon off (b) Lactose present, repressor inactive, operon on 5′ 3 DNA lac operon RNA polymerase Start codon Stop codon mRNA 3′ Protein Protein Inactive repressorAllolactose (inducer) mRNA 5′ Permease Transacetylaseβ-Galactosidase l a cI l a cI
- 9. • Inducible enzymesusually function in catabolic pathways; their synthesis is induced by a chemical signal • Repressible enzymes usually function in anabolic pathways; their synthesis is repressed by high levels of the end product • Regulation of the trp and lac operons involves negative control of genes because operons are switched off by the active form of the repressor
- 10. Positive Gene Regulation •Some operons are also subject to positive control through a stimulatory protein, such as catabolite activator protein (CAP), an activator of transcription • When glucose (a preferred food source of E. coli) is scarce, CAP is activated by binding with cyclic AMP • Activated CAP attaches to the promoter of the lac operon and increases the affinity of RNA polymerase, thus accelerating transcription
- 11. Figure 18.5 Promoter DNA Operator Promoter DNA CAP-binding site cAMP Active CAP Inactive CAP RNA polymerase bindsand transcribes lac I lac I Allolactose Inactive lac repressor (a) Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized lacZ lacZ CAP-binding site RNA polymerase less likely to bind Operator Inactive CAP Inactive lac repressor (b) Lactose present, glucose present (cAMP level low): little lac mRNA synthesized
- 12. Eukaryotic gene expression:Differential Gene Expression • Almost all the cells in an organism are genetically identical • Differences between cell types result from differential gene expression, the expression of different genes by cells with the same genome • Errors in gene expression can lead to diseases including cancer • Gene expression is regulated at many stages
- 13. Figure 18.6 Signal Chromatin DNA Geneavailable for transcription RNA Exon Intron Cap Primary transcript Tail mRNA in nucleus NUCLEUS Transcription RNA processing Transport to cytoplasm Chromatin modification: DNA unpacking CYTOPLASM mRNA in cytoplasm TranslationDegradation of mRNA Polypeptide Protein processing Active protein Degradation of protein Transport to cellular destination Cellular function (such as enzymatic activity or structural support) Many places to regulate: review examples at each box with your neighbor
- 14. Regulation of ChromatinStructure • Genes within highly packed heterochromatin are usually not expressed e.g. Barr Body • Chemical modifications to histones and DNA of chromatin influence both chromatin structure and gene expression
- 15. Overview: How EukaryoticGenomes Work and Evolve • Two features of eukaryotic genomes are a major information-processing challenge: – First, the typical eukaryotic genome is much larger than that of a prokaryotic cell – Second, cell specialization limits the expression of many genes to specific cells • The DNA-protein complex, called chromatin, is ordered into higher structural levels than the DNA- protein complex in prokaryotes
- 16. LE 19-2a DNA doublehelix Histone tails His- tones Linker DNA (“string”) Nucleosome (“bead”) 10 nm 2 nm Histone H1 Nucleosomes (10-nm fiber) Level 1
- 17. LE 19-2b 30 nm Nucleosome 30-nmfiber The next level of packing forms the 30-nm chromatin fiber Level 2
- 18. LE 19-2c 300 nm Loops Scaffold Proteinscaffold Looped domains (300-nm fiber) In turn, the 30-nm fiber forms looped domains, making up a 300-nm fiber Level 3
- 19. LE 19-2d Metaphase chromosome 700nm 1,400 nm In a mitotic chromosome, the looped domains coil and fold, forming the metaphase chromosome Level 4 Level 5
- 20. • Interphase chromatinis usually much less condensed than that of mitotic chromosomes • Much of the interphase chromatin is present as a 10-nm fiber, and some is 30-nm fiber, which in some regions is folded into looped domains • Interphase chromosomes have highly condensed areas, called heterochromatin, and less compacted areas, called euchromatin
- 21. Histone Modifications • Inhistone acetylation, acetyl groups are attached to positively charged lysines in histone tails • This process loosens chromatin structure, thereby promoting the initiation of transcription • The addition of methyl groups (methylation) can condense chromatin; the addition of phosphate groups (phosphorylation) next to a methylated amino acid can loosen chromatin
- 22. Fig. 18-7 Histone tails DNA double helix (a)Histone tails protrude outward from a nucleosome Acetylated histones Amino acids available for chemical modification (b) Acetylation of histone tails promotes loose chromatin structure that permits transcription Unacetylated histones
- 23. DNA Methylation • DNAmethylation, the addition of methyl groups to certain bases in DNA, is associated with reduced transcription in some species • DNA methylation can cause long-term inactivation of genes in cellular differentiation • In genomic imprinting, methylation regulates expression of either the maternal or paternal alleles of certain genes at the start of development When passed to next generation, it is called??
- 24. DNA Methylation • DNAmethylation, the addition of methyl groups to certain bases in DNA, is associated with reduced transcription in some species • DNA methylation can cause long-term inactivation of genes in cellular differentiation • In genomic imprinting, methylation regulates expression of either the maternal or paternal alleles of certain genes at the start of development When passed to next generation, it is called?? Epigenetic
- 25. Organization of aTypical Eukaryotic Gene • Associated with most eukaryotic genes are control elements, segments of noncoding DNA that help regulate transcription by binding certain proteins • Control elements and the proteins they bind are critical to the precise regulation of gene expression in different cell types
- 26. Figure 18.8 Enhancer (groupof distal control elements) Proximal control elements Transcription start site Promoter Exon ExonPrimary RNA transcript (pre-mRNA) Intron Intron Exon Exon Intron Intron Exon Exon Poly-A signal sequence Transcription termination region Downstream Poly-A signal Cleaved 3′ end of primary transcript 5′ 3′ Transcription Upstream DNA Intron RNA RNA processing Coding segment Start codon Stop codon 3′ UTR Poly-A tail AAA⋯AAAmRNA 5′ Cap 5′ UTR G P P P
- 27. The Roles ofTranscription Factors • To initiate transcription, eukaryotic RNA polymerase requires the assistance of proteins called transcription factors • General transcription factors are essential for the transcription of all protein-coding genes and bind to the promoter region • In eukaryotes, high levels of transcription of particular genes depend on control elements interacting with specific transcription factors
- 28. • Proximal controlelements are located close to the promoter • Distal control elements, groups of which are called enhancers, may be far away from a gene or even located in an intron Enhancers and Specific Transcription Factors
- 29. Figure 18.10-3 DNA ActivatorsPromoter Enhancer Distal control element TATA box Gene DNA- bending protein Group of mediator proteins General transcription factors RNA polymerase II RNA polymerase II RNA synthesis Transcription initiation complex
- 30. • An activatoris a protein that binds to an enhancer and stimulates transcription of a gene • Bound activators cause mediator proteins to interact with proteins at the promoter Combinatorial Control of Gene Activation • A particular combination of control elements can activate transcription only when the appropriate activator proteins are present
- 31. Fig. 18-10 Control elements Enhancer Available activators Albumin gene (b)Lens cell Crystallin gene expressed Available activators LENS CELL NUCLEUS LIVER CELL NUCLEUS Crystallin gene Promoter (a) Liver cell Crystallin gene not expressed Albumin gene expressed Albumin gene not expressed
- 32. • Some transcriptionfactors function as repressors, inhibiting expression of a particular gene • Some activators and repressors act indirectly by influencing chromatin structure to promote or silence transcription
- 33. Coordinately Controlled Genesin Eukaryotes • Unlike the genes of a prokaryotic operon, each of the coordinately controlled eukaryotic genes has a promoter and control elements • These genes can be scattered over different chromosomes, but each has the same combination of control elements • Copies of the activators recognize specific control elements and promote simultaneous transcription of the genes
- 34. Nuclear Architecture andGene Expression • Loops of chromatin extend from individual chromosome territories into specific sites in the nucleus • Loops from different chromosomes may congregate at particular sites, some of which are rich in transcription factors and RNA polymerases • These may be areas specialized for a common function
- 35. Exons RNA splicing 1 23 4 5 1 2 3 4 5 1 2 3 5 1 2 4 5OR Troponin T gene DNA Primary RNA transcript mRNA alternative RNA splicing, different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns
- 36. RNA turnover Protein turnover(shown here) Proteasome and ubiquitin to be recycledProteasome Protein fragments (peptides)Protein entering a proteasome Ubiquitinated protein Protein to be degraded Ubiquitin
- 37. Concept 18.3: NoncodingRNAs play multiple roles in controlling gene expression • Only a small fraction of DNA codes for proteins, rRNA, and tRNA • A significant amount of the genome may be transcribed into noncoding RNAs • Noncoding RNAs regulate gene expression at two points: mRNA translation and chromatin configuration
- 38. Effects on mRNAsby MicroRNAs and Small Interfering RNAs • MicroRNAs (miRNAs) are small single- stranded RNA molecules that can bind to mRNA • These can degrade mRNA or block its translation
- 39. Figure 18.14 alsocalled siRNA for silencing RNAor small interfering RNA miRNA miRNA- protein complex The miRNA binds to a target mRNA. mRNA degraded Translation blocked OR If bases are completely complementary, mRNA is degraded. If match is less than complete, translation is blocked. 1 2
- 40. • The phenomenonof inhibition of gene expression by RNA molecules is called RNA interference (RNAi) • RNAi is caused by small interfering RNAs (siRNAs) • RNAi is used in the laboratory as a means of disabling genes to investigate their function • siRNAs and miRNAs are similar but form from different RNA precursors
- 41. Chromatin Remodeling andSilencing of Transcription by Small RNAs • siRNAs play a role in heterochromatin formation and can block large regions of the chromosome • Small RNAs may also block transcription of specific genes
- 42. Figure 18.15. Insome yeasts siRNAs re-form heterochromatin at centromeres after chromosome replication RNA transcripts (red) produced. Yeast enzyme synthesizes strands complementary to RNA transcripts. Double-stranded RNA processed into siRNAs that associate with proteins. The siRNA-protein complexes recruit histone-modifying enzymes. The siRNA-protein complexes bind RNA transcripts and become tethered to centromere region. Chromatin condensation is initiated and heterochromatin is formed. 1 2 3 4 5 6 Centromeric DNA RNA polymerase RNA transcript Sister chromatids (two DNA molecules) siRNA-protein complex Centromeric DNA Chromatin- modifying enzymes Heterochromatin at the centromere region
- 43. Fig. 18-UN5 Chromatin modification RNAprocessing TranslationmRNA degradation Protein processing and degradation mRNA degradation • miRNA or siRNA can target specific mRNAs for destruction. • miRNA or siRNA can block the translation of specific mRNAs. Transcription • Small RNAs can promote the formation of heterochromatin in certain regions, blocking transcription. Chromatin modification Translation
- 44. Summary • Genes inhighly compacted chromatin are generally not transcribed. Chromatin modification • DNA methylation generally reduces transcription. • Histone acetylation seems to loosen chromatin structure, enhancing transcription. Chromatin modification Transcription RNA processing TranslationmRNA degradation Protein processing and degradation mRNA degradation • Each mRNA has a characteristic life span, determined in part by sequences in the 5 and 3 UTRs. • Protein processing and degradation by proteasomes are subject to regulation. Protein processing and degradation • Initiation of translation can be controlled via regulation of initiation factors. Translation ormRNA Primary RNA transcript • Alternative RNA splicing: RNA processing • Coordinate regulation: Enhancer for liver-specific genes Enhancer for lens-specific genes Bending of the DNA enables activators to contact proteins at the promoter, initiating transcription. Transcription • Regulation of transcription initiation: DNA control elements bind specific transcription factors.
- 45. Concept 18.4: Aprogram of differential gene expression leads to the different cell types in a multicellular organism • During embryonic development, a fertilized egg gives rise to many different cell types • Cell types are organized successively into tissues, organs, organ systems, and the whole organism • Gene expression orchestrates the developmental programs of animals
- 46. • Cell differentiationis the process by which cells become specialized in structure and function • The physical processes that give an organism its shape constitute morphogenesis • Differential gene expression results from genes being regulated differently in each cell type • Materials in the egg can set up gene regulation that is carried out as cells divide
- 47. Concept 18.5: Cancerresults from genetic changes that affect cell cycle control • The gene regulation systems that go wrong during cancer are the very same systems involved in embryonic development • Cancer can be caused by mutations to genes that regulate cell growth and division • Tumor viruses can cause cancer in animals including humans
- 48. Oncogenes and Proto-Oncogenes •Oncogenes are cancer-causing genes • Proto-oncogenes are the corresponding normal cellular genes that are responsible for normal cell growth and division • Conversion of a proto-oncogene to an oncogene can lead to abnormal stimulation of the cell cycle
- 49. • Proto-oncogenes canbe converted to oncogenes by – Movement of DNA within the genome: if it ends up near an active promoter, transcription may increase – Amplification of a proto-oncogene: increases the number of copies of the gene – Point mutations in the proto-oncogene or its control elements: cause an increase in gene expression
- 50. Figure 18.23 Proto-oncogene Proto-oncogeneProto-oncogene Point mutation:Gene amplification: multiple copies of the gene Translocation or transposition: gene moved to new locus, under new controls New promoter Oncogene Oncogene Oncogene within the gene within a control element Normal growth- stimulating protein in excess Normal growth-stimulating protein in excess Normal growth- stimulating protein in excess Hyperactive or degradation- resistant protein
- 51. Tumor-Suppressor Genes • Tumor-suppressorgenes help prevent uncontrolled cell growth • Mutations that decrease protein products of tumor-suppressor genes may contribute to cancer onset • Tumor-suppressor proteins – Repair damaged DNA – Control cell adhesion – Inhibit the cell cycle in the cell-signaling pathway
- 52. Interference with NormalCell-Signaling Pathways • Mutations in the ras proto-oncogene and p53 tumor-suppressor gene are common in human cancers • Mutations in the ras gene can lead to production of a hyperactive Ras protein and increased cell division
- 53. Figure 18.24 G protein Growthfactor Receptor Protein kinases Transcription factor (activator) NUCLEUS Protein that stimulates the cell cycle Transcription factor (activator) NUCLEUS Overexpression of protein Ras Ras MUTATION GTP GTP Ras protein active with or without growth factor. P P P P P P 1 3 2 5 4 6
- 54. • Suppression ofthe cell cycle can be important in the case of damage to a cell’s DNA; p53 prevents a cell from passing on mutations due to DNA damage • Mutations in the p53 gene prevent suppression of the cell cycle
- 55. Figure 18.25 Protein kinases DNAdamage in genome Active form of p53 Transcription DNA damage in genome UV light UV light Defective or missing transcription factor. Inhibitory protein absent Protein that inhibits the cell cycleNUCLEUS MUTATION 1 3 4 2 5
- 56. The Multistep Modelof Cancer Development • Multiple mutations are generally needed for full-fledged cancer; thus the 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
- 57. Figure 18.26a 1 Colon wall Lossof tumor- suppressor gene APC (or other) Normal colon epithelial cells 2 3 4 5 Activation of ras oncogene Additional mutations Loss of tumor- suppressor gene SMAD4 Larger benign growth (adenoma) Malignant tumor (carcinoma) Small benign growth (polyp) Loss of tumor-suppressor gene p53
- 58. Inherited Predisposition andOther Factors Contributing to Cancer • Individuals can inherit oncogenes or mutant alleles of tumor-suppressor genes • Inherited mutations in the tumor-suppressor gene adenomatous polyposis coli are common in individuals with colorectal cancer • Mutations in the BRCA1 or BRCA2 gene are found in at least half of inherited breast cancers
- 59. You should nowbe able to: 1. Explain the concept of an operon and the function of the operator, repressor, and corepressor 2. Explain the adaptive advantage of grouping bacterial genes into an operon 3. Explain how repressible and inducible operons differ and how those differences reflect differences in the pathways they control 4. Explain how DNA methylation and histone acetylation affect chromatin structure and the regulation of transcription 5. Define control elements and explain how they influence transcription in eukaryotes 6. Explain the role of promoters, enhancers, activators, and repressors in transcription control 7. Explain how eukaryotic genes can be coordinately expressed 8. Describe the roles played by small RNAs on gene expression 9. Describe two sources of information that instruct a cell to express genes at the appropriate time 10. Explain how mutations in tumor-suppressor genes can contribute to cancer 11. Describe the effects of mutations to the p53 and ras genes