1. Chapter 18 Regulation of Gene Expression

2. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Overview: Conducting the Genetic Orchestra 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

3. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Concept 18.1: Bacteria often respond to environmental change by regulating transcription Natural selection has favored bacteria that produce only the products needed by that cell A cell can regulate the production of enzymes by feedback inhibition or by gene regulation Gene expression in bacteria is controlled by the operon model

4. Fig. 18-2 Regulation of gene expression trpE gene trpD gene trpC gene trpB gene trpA gene (b) Regulation of enzyme production (a) Regulation of enzyme activity Enzyme 1 Enzyme 2 Enzyme 3 Tryptophan Precursor Feedback inhibition

5. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings I. Operons Functionally related genes can be coordinated by a single on-off “switch” – segment of DNA called an operator usually positioned within the promoter (ex. TATA box) Operon = the operator, the promoter, and the genes that they control

6. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Repressor - protein that can switch off operon – prevents gene transcription by binding to the operator and blocking RNA polymerase – is the product of a separate regulatory gene – can be active or inactive form, depending on the presence of other molecules

7. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Corepressor = molecule that cooperates with a repressor protein to switch an operon off Ex. E. coli uses the amino acid tryptophan

8. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings trp operon is on and the genes for tryptophan synthesis are transcribed When tryptophan is present, it binds to the trp repressor protein, which turns the operon off Repressor is active in the presence of its corepressor tryptophan; thus the trp operon is turned off (repressed) if tryptophan levels are high

9. Fig. 18-3 Polypeptide subunits that make up enzymes for tryptophan synthesis (b) Tryptophan present, repressor active, operon off Tryptophan (corepressor) (a) Tryptophan absent, repressor inactive, operon on No RNA made Active repressor mRNA Protein DNA mRNA 5 Protein Inactive repressor RNA polymerase Regulatory gene Promoter trp operon Genes of operon Operator Stop codon Start codon mRNA trpA 5 3 trpR trpE trpD trpC trpB ABC D E

10. Fig. 18-3a Polypeptide subunits that make up enzymes for tryptophan synthesis (a) Tryptophan absent, repressor inactive, operon on DNA mRNA 5 ProteinInactive repressor RNA polymerase Regulatory gene Promoter trp operon Genes of operon Operator Stop codon Start codon mRNA trpA 5 3 trpRtrpE trpD trpCtrpB AB CD E

11. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings II. Negative Gene Regulation Repressible operon is one that is usually on; binding of a repressor to the operator shuts off transcription (trp operon)

12. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Inducible operon is one that is usually off; a molecule called an inducer inactivates the repressor and turns on transcription – Ex. lac operon contains genes that code for enzymes used in hydrolysis of lactose – The lac repressor is active and switches the lac operon off – Inducer – a molecule that inactivates the repressor to turn the lac operon on

13. Fig. 18-4 (b) Lactose present, repressor inactive, operon on (a) Lactose absent, repressor active, operon off mRNA Protein DNA mRNA 5 Protein Active repressor RNA polymerase Regulatory gene Promoter Operator mRNA 5 3 Inactive repressor Allolactose (inducer) 5 3 No RNA made RNA polymerase Permease Transacetylase lac operon  -Galactosidase lacY lacZ lacAlac I lacZ

14. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Inducible enzymes function in catabolic (breakdown) pathways; synthesis is induced by a chemical signal Repressible enzymes usually function in anabolic (building) pathways; synthesis is repressed by high levels of the end product Negative control of genes = operons are switched off by the active form of the repressor

15. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings III. Positive Gene Regulation Operons can have positive control through a stimulatory protein, ex. catabolite activator protein (CAP), an activator of transcription When glucose (main 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, making more lactase

16. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings When glucose levels increase, CAP detaches from the lac operon, and transcription returns to a normal rate CAP helps regulate other operons that encode enzymes used in catabolic pathways

17. Fig. 18-5 (b) Lactose present, glucose present (cAMP level low): little lac mRNA synthesized cAMP DNA Inactive lac repressor Allolactose Inactive CAP lac I CAP-binding site Promoter Active CAP Operator lacZ RNA polymerase binds and transcribes Inactive lac repressor lacZ Operator Promoter DNA CAP-binding site lac I RNA polymerase less likely to bind Inactive CAP (a) Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized

18. Gene Regulation - Andersen 10min

19. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Concept 18.2: Eukaryotic gene expression can be regulated at any stage All organisms must regulate which genes are expressed at any given time In multicellular organisms gene expression is essential for cell specialization

20. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings IV. Differential Gene Expression Almost all cells in an organism are genetically identical Differences between cell types result from differential gene expression (expression of different genes by cells with same genome) Errors in gene expression can lead to diseases including cancer

21. Fig. 18-6 DNA Signal Gene NUCLEUS Chromatin modification Chromatin Gene available for transcription Exon Intron Tail RNA Cap RNA processing Primary transcript mRNA in nucleus Transport to cytoplasm mRNA in cytoplasm Translatio n CYTOPLASM Degradation of mRNA Protein processing Polypeptide Active protein Cellular function Transport to cellular destination Degradation of protein Transcription

22. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings V. Regulation of Chromatin Structure Genes within highly packed heterochromatin are not usually expressed

23. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings A. Histone Modifications Histone acetylation = acetyl groups (-COCH 3 ) are attached to lysines in histone tails (charge neutralized), histones no longer bind to other nucleosomes Loosens chromatin structure, promoting the start of transcription Adding methyl groups (methylation) can condense chromatin; adding phosphate groups (phosphorylation) can loosen chromatin

24. 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

25. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings B. DNA Methylation DNA methylation (addition of methyl groups to certain bases) results is reduced transcription Can cause long-term inactivation of genes in cellular differentiation Genomic imprinting, methylation regulates expression of either the maternal or paternal alleles of certain genes at the start of development

26. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings C. Epigenetic Inheritance These modifications may be passed to future generations of cells Epigenetic inheritance - inheritance of traits transmitted by mechanisms not involving nucleotide sequence

27. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings VI. Regulation of Transcription Initiation Chromatin-modifying enzymes provide initial control of gene expression (makes a region of DNA more / less able to bind the transcription factors

28. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings A. Organization of Eukaryotic Genes Control elements = segments of noncoding DNA that regulate transcription by binding certain proteins – critical to precise regulation of gene expression in different cell types

29. Fig. 18-8-3 Enhancer (distal control elements) Proximal control elements Poly-A signal sequence Termination region Downstream Promoter Upstream DNA Exon Intron Exon Intron Cleaved 3 end of primary transcript Primary RNA transcript Poly-A signal Transcription 5 RNA processing Intron RNA Coding segment mRNA 5 Cap 5 UTR Start codon Stop codon 3 UTR Poly-A tail 3

30. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings B. Roles of Transcription Factors Eukaryotic RNA polymerase needs transcription factors Enhancers and Specific Transcription Factors – Proximal control elements = close to promoter – Distal control elements (enhancers) = far away from a gene / located in an intron

31. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Activator = protein that binds to an enhancer and stimulates transcription Bound activators cause mediator proteins to interact with proteins at the promoter Some TFs function as repressors, inhibiting a gene

32. Fig. 18-9-3 Enhancer TATA box Promoter Activators DNA Gene Distal control element Group of mediator proteins DNA-bending protein General transcription factors RNA polymerase II RNA polymerase II Transcription initiation complex RNA synthesis

33. 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

34. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings VII. Post-Transcriptional Regulation Regulatory mechanisms can operate at various stages after transcription, allow a cell to rapidly express a gene in response to env changes

35. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings A. RNA Processing Alternative RNA splicing = different mRNA molecules are produced from the same transcript, depending on which RNA segments are treated as exons and which as introns

36. Fig. 18-11 or RNA splicing mRNA Primary RNA transcript Troponin T gene Exons DNA

37. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings B. mRNA Degradation Eukaryotic mRNA is more long lived than prokaryotic mRNA (pro can respond to changes faster) mRNA life span is determined in part by sequences in the leader and trailer regions

38. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings D. Protein Processing and Degradation Proteasomes are giant protein complexes that bind protein molecules and degrade them Proteasome and ubiquitin to be recycled Proteasome Protein fragments (peptides) Protein entering a proteasome Ubiquitinated protein Protein to be degraded Ubiquitin

39. Fig. 18-UN4 Genes in highly 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 Translation mRNA 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.

40. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Concept 18.3: Noncoding RNAs 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

41. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings VIII. MicroRNAs and Small Interfering RNAs MicroRNAs (miRNAs) = small single-stranded RNA molecules that can bind to mRNA – Degrade mRNA or block its translation

42. Fig. 18-13 miRNA- protein complex (a) Primary miRNA transcript Translation blocked Hydrogen bond (b) Generation and function of miRNAs Hairpin miRNA Dicer 3 mRNA degraded 5

43. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings RNA interference (RNAi) = inhibition of gene expression by RNA molecules siRNAs and miRNAs are similar but form from different RNA precursors

44. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Concept 18.4: A program 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

45. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings IX. Embryonic Development Cell differentiation = cells become specialized in structure and function Morphogenesis = physical processes that give an organism its shape 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

46. Fig. 18-14 (a) Fertilized eggs of a frog (b) Newly hatched tadpole

47. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings X. Cytoplasmic Determinants and Inductive Signals Egg’s cytoplasm contains RNA, proteins, and other substances that are distributed unevenly in the unfertilized egg Cytoplasmic determinants = maternal substances in the egg that influence early development As zygote divides by mitosis, cells contain different cytoplasmic determinants, which lead to different gene expression

48. Fig. 18-15a (a) Cytoplasmic determinants in the egg Two different cytoplasmic determinants Unfertilized egg cell Sperm Fertilization Zygote Mitotic cell division Two-celled embryo Nucleus

49. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Induction = signal molecules from embryonic cells cause transcriptional changes in nearby target cells – interactions between cells induce differentiation of specialized cell types

50. Fig. 18-15b (b) Induction by nearby cells Signal molecule (inducer) Signal transduction pathway Early embryo (32 cells) NUCLEUS Signal receptor

51. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings XI. Cellular Differentiation Determination commits a cell to its final fate Determination precedes differentiation Cell differentiation is marked by the production of tissue-specific proteins

52. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Myoblasts produce muscle-specific proteins and form skeletal muscle cells MyoD is one of several “master regulatory genes” that produce proteins that commit the cell to becoming skeletal muscle The MyoD protein is a transcription factor that binds to enhancers of various target genes

53. Fig. 18-16-3 Embryonic precursor cell Nucleus OFF DNA Master regulatory gene myoD Other muscle-specific genes OFF mRNA MyoD protein (transcription factor) Myoblast (determined) mRNA Myosin, other muscle proteins, and cell cycle– blocking proteins Part of a muscle fiber (fully differentiated cell) MyoD Another transcription factor

54. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Concept 18.5: Cancer results 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

55. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings XII. Genes Associated with Cancer Cancer can be caused by mutations to genes that regulate cell growth and division Tumor viruses can cause cancer in animals including humans

56. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings A. Oncogenes and Proto-Oncogenes Oncogenes = cancer-causing genes Proto-oncogenes = the corresponding normal cellular genes (responsible for normal growth and division) Conversion of proto-oncogene to oncogene = abnormal stimulation of the cell cycle

57. Fig. 18-20 Normal growth- stimulating protein in excess New promoter DNA Proto-oncogene Gene amplification: Translocation or transposition: Normal growth-stimulating protein in excess Normal growth- stimulating protein in excess Hyperactive or degradation- resistant protein Point mutation: Oncogene within a control element within the gene

58. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Conversion to oncogenes by – Movement of DNA w/in genome: if it ends up near an active promoter, transcription may increase – Amplification: increases the number of copies of the gene – Point mutations in the proto-oncogene or its control elements: causes an increase in gene expression

59. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings B. Tumor-Suppressor Genes Tumor-suppressor genes = help prevent uncontrolled cell growth – Mutations that decrease proteins from 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

60. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings XIII. Interference with Normal Cell-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

61. Fig. 18-21a Receptor Growth factor G protein GTP Ras GTP Ras Protein kinases (phosphorylation cascade) Transcription factor (activator) DNA Hyperactive Ras protein (product of oncogene) issues signals on its own MUTATION NUCLEUS Gene expression Protein that stimulates the cell cycle (a) Cell cycle–stimulating pathway 1 1 3 4 5 2

62. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Suppression of the 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

63. Fig. 18-21b MUTATION Protein kinases DNA DNA damage in genome Defective or missing transcription factor, such as p53, cannot activate transcription Protein that inhibits the cell cycle Active form of p53 UV light (b) Cell cycle–inhibiting pathway 2 3 1

64. Fig. 18-21c (c) Effects of mutations EFFECTS OF MUTATIONS Cell cycle not inhibited Protein absent Increased cell division Protein overexpressed Cell cycle overstimulated

65. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings XIV. Multistep Model of Cancer Development Multiple mutations are generally needed for full-fledged cancer; thus the incidence increases with age A cancerous cell is usually characterized by at least one active oncogene and the mutation of several tumor-suppressor genes

66. Fig. 18-22a Colon Colon wall Normal colon epithelial cells

67. Fig. 18-22b Loss of tumor- suppressor gene APC (or other) Small benign growth (polyp) 1

68. Fig. 18-22c Activation of ras oncogene Loss of tumor-suppressor gene DCC Larger benign growth (adenoma) 2 3

69. Fig. 18-22d Malignant tumor (carcinoma) Loss of tumor-suppressor gene p53 Additional mutations 5 4

70. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings You should now be 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

71. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 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 6.Explain the role of promoters, enhancers, activators, and repressors in transcription control

72. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 7.Explain how eukaryotic genes can be coordinately expressed 8.Describe the roles played by small RNAs on gene expression 9.Explain why determination precedes differentiation 10.Describe two sources of information that instruct a cell to express genes at the appropriate time

73. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 11.Explain how maternal effect genes affect polarity and development in Drosophila embryos 12.Explain how mutations in tumor-suppressor genes can contribute to cancer 13.Describe the effects of mutations to the p53 and ras genes