1. LECTURE PRESENTATIONS For CAMPBELL BIOLOGY, NINTH EDITION Jane B. Reece, Lisa A. Urry, Michael L. Cain, Steven A. Wasserman, Peter V. Minorsky, Robert B. Jackson © 2011 Pearson Education, Inc. Lectures by Erin Barley Kathleen Fitzpatrick Regulation of Gene Expression Chapter 18

2. Overview: Conducting the Genetic Orchestra Cells intricately and precisely regulate their gene 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 In this chapter we explore –How bacteria and Eukaryotes regulate expression of their genes RNA molecules play many roles in regulating gene expression in eukaryotes © 2011 Pearson Education, Inc.

3. 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 –Bacteria cells that conserve resources and energy have a selective advantage over cells that are unable to do so A cell can regulate the production of enzymes by feedback inhibition or by gene regulation –E.g. In an environment lacking the amino acid tryptophan, E. coli activates a metabolic pathway to synthesize tryptophan –In the presence of tryptophan the bacterial cell stops producing tryptophan © 2011 Pearson Education, Inc.

4. Metabolic control can take place in two levels 1.Cell can adjust the activity of enzymes already present - Such feed back inhibition is typical of anabolic pathways 2.Cells can adjust the production level of certain enzymes; regulate the expression of the genes encoding the enzymes 1.Control of enzyme production occurs at the level of trascription. Gene expression in bacteria is controlled by the operon model

5. Precursor Feedback inhibition Enzyme 1 Enzyme 2 Enzyme 3 Tryptophan (a) (b) Regulation of enzyme activity Regulation of enzyme production Regulation of gene expression   trpE gene trpD gene trpC gene trpB gene trpA gene Figure 18.2

6. Operons: The Basic Concept A cluster of functionally related genes can be under coordinated control by a single “on-off switch” –E.g. Grouping genes of related function into one transcription unit –E coli synthesizes amino acid tryptophan from the precursor molecule in a multistep pathway; tryp operon 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 – operator –the promoter –and the genes that they control © 2011 Pearson Education, Inc.

7. How does the operator (operon switch control transcription) –By itself the operon is turned on thus RNA polymerase can bind and initiate trascription –The operon can be switched off by a protein called a repressor E.g. The trp operon can be turned off by the trp repressor The repressor prevents gene transcription by binding to the operator and blocking RNA polymerase A repressor is specific for the operator of a particular operon and is the product of a separate regulatory gene (trpR) © 2011 Pearson Education, Inc.

8. The repressor can be in an active or inactive form (allosteric), depending on the presence of other molecules –Tryp repressor is synthesized in an inactive form. Binding of tryptophan to tryp repressor activates it, thus can bind to operator and turn it off –Binding of repressors is also reversible –An operator can switch between two states One without the repressor bound One with the repressor bound A corepressor is a molecule that cooperates with a repressor protein to switch an operon off –For example, E. coli can synthesize the amino acid tryptophan. As tryptophan accumulates, more tryptophan molecules assist the repressor to shut down production © 2011 Pearson Education, Inc.

9. By default the 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 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 © 2011 Pearson Education, Inc.

10. Figure 18.3a Promoter DNA Regulatory gene mRNA trpR 5 3 Protein Inactive repressor RNA polymerase Promoter trp operon Genes of operon Operator mRNA 5 Start codonStop codon trpEtrpDtrpC trpB trpA EDCBA Polypeptide subunits that make up enzymes for tryptophan synthesis (a) Tryptophan absent, repressor inactive, operon on

11. Figure 18.3b-1 (b) Tryptophan present, repressor active, operon off DNA mRNA Protein Tryptophan (corepressor) Active repressor

12. Figure 18.3b-2 (b) Tryptophan present, repressor active, operon off DNA mRNA Protein Tryptophan (corepressor) Active repressor No RNA made

13. Repressible and Inducible Operons: Two Types of Negative Gene Regulation Two types of operons –Repressible operons –Inducible operons A repressible operon is one that is usually on – binding of a repressor to the operator shuts off transcription –Enzymes for tryptophan synthesis are repressible 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 –Enzymes of the lactose pathway are called inducible enzymes (snthesis is induced by allolactose) nzymes © 2011 Pearson Education, Inc.

14. The lac operon is an inducible operon and contains genes that code for enzymes used in the hydrolysis and metabolism of lactose –lacI is the regulatory gene; located outside the operon 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 –For the lac operon, the inducer is allolactose, an isomer of lactose formed from small amounts of lactose –In the absence of lactose (allolactose) the represor is active and the genes of the lac operon are siliced –In the presence of lactose, allolactose binds to the lac repressor, changes it’s conformation preventing it from attaching to the operator © 2011 Pearson Education, Inc.

15. Figure 18.4a (a) Lactose absent, repressor active, operon off Regulatory gene Promoter Operator DNA lacZ lac I DNA mRNA 5 3 No RNA made RNA polymerase Active repressor Protein

16. Figure 18.4b (b) Lactose present, repressor inactive, operon on lac I lac operon lacZlacYlacADNA mRNA 5 3 Protein mRNA 5 Inactive repressor RNA polymerase Allolactose (inducer)  -Galactosidase PermeaseTransacetylase

17. Inducible enzymes usually 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 © 2011 Pearson Education, Inc.

18. 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 (binds to DNA activates transcription) When glucose (a preferred food source of E. coli) is scarce, CAP is activated by binding with cyclic AMP (cAMP) –cAMP accumulates when glucose is scarce Activated CAP attaches to the promoter of the lac operon and increases the affinity of RNA polymerase, thus accelerating transcription © 2011 Pearson Education, Inc.

19. 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 Thus the lac operon is under dual control –Negative control by the lac repressor –Positive control by CAP © 2011 Pearson Education, Inc.

20. Figure 18.5a Promoter DNA CAP-binding site lacZ lac I RNA polymerase binds and transcribes Operator cAMP Active CAP Inactive CAP Allolactose Inactive lac repressor (a) Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized

21. Figure 18.5b Promoter DNA CAP-binding site lacZ lac I Operator RNA polymerase less likely to bind Inactive lac repressor Inactive CAP (b) Lactose present, glucose present (cAMP level low): little lac mRNA synthesized

22. Concept 18.2: Eukaryotic gene expression is regulated at many stages All organisms must regulate which genes are expressed at any given time –They must continually turn genes on and off in response to external stimuli/signals In multicellular organisms regulation of gene expression is essential for cell specialization © 2011 Pearson Education, Inc.

23. Differential Gene Expression Almost all the cells in an organism are genetically identical Muscles and nerve cells express smaller fractions of their genes Differences between cell types result from differential gene expression – the expression of different genes by cells with the same genome The function of any cell depends on the appropriate sets of genes being expressed Transcription factors locate the right genes at the right time (it’s like looking for a needle in a haystack) Abnormalities in gene expression can lead to diseases including cancer Gene expression is regulated at many stages –In all organisms transcription is a common control point © 2011 Pearson Education, Inc.

24. Figure 18.6 Signal NUCLEUS Chromatin Chromatin modification: DNA unpacking involving histone acetylation and DNA demethylation DNA Gene Gene available for transcription RNA Exon Primary transcript Transcription Intron RNA processing Cap Tail mRNA in nucleus Transport to cytoplasm CYTOPLASM mRNA in cytoplasm Translation Degradation of mRNA Polypeptide Protein processing, such as cleavage and chemical modification Active protein Degradation of protein Transport to cellular destination Cellular function (such as enzymatic activity, structural support)

25. Regulation of Chromatin Structure DNA of Eukaryotic cells is packaged in chromatin Genes within highly packed heterochromatin are usually not expressed Chemical modifications to histones and DNA of chromatin influence both chromatin structure and gene expression © 2011 Pearson Education, Inc.

26. Histone Modifications In histone acetylation, acetyl groups are attached to positively charged lysines in histone tail –Acetylation promotes initiation of transcription. Deacetylation does not This 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 © 2011 Pearson Education, Inc. Animation: DNA Packing

27. Figure 18.7 Amino acids available for chemical modification Histone tails DNA double helix Nucleosome (end view) (a) Histone tails protrude outward from a nucleosome Unacetylated histones Acetylated histones (b) Acetylation of histone tails promotes loose chromatin structure that permits transcription

28. The histone code hypothesis proposes that specific combinations of modifications, as well as the order in which they occur, help determine chromatin configuration and influence transcription © 2011 Pearson Education, Inc.

29. DNA Methylation DNA methylation, 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 © 2011 Pearson Education, Inc.

30. Regulation of Transcription Initiation Chromatin-modifying enzymes provide initial control of gene expression by making a region of DNA either more or less able to bind the transcription machinery Once the chromatin of a gene is optimally modified for expression, transcription is the next major step where gene expression is regulated © 2011 Pearson Education, Inc.

31. Organization of a Typical Eukaryotic Gene Associated with most eukaryotic genes are multiple control elements, segments of noncoding DNA that serve as binding sites for transcription factors that help regulate transcription Control elements and the transcription factors they bind are critical to the precise regulation of gene expression in different cell types © 2011 Pearson Education, Inc.

32. The Roles of Transcription 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 In eukaryotes, high levels of transcription of particular genes depend on control elements interacting with specific transcription factors © 2011 Pearson Education, Inc.

33. Proximal control elements are located close to the promoter Distal control elements, groupings of which are called enhancers, may be far away from a gene or even located in an intron –The functioning of enhancers is an example of transcriptional control of gene expression In Eukaryotes, the rate of gene expression can be strongly increased or decreased by the binding of specific trasncription factors either activators or represors Enhancers and Specific Transcription Factors © 2011 Pearson Education, Inc.

34. An activator is a protein that binds to an enhancer and stimulates transcription of a gene Activators have two domains, one that binds DNA and a second that activates transcription Bound activators facilitate a sequence of protein- protein interactions that result in transcription of a given gene © 2011 Pearson Education, Inc. Animation: Initiation of Transcription

35. Figure 18.8-1 Enhancer (distal control elements) DNA Upstream Promoter Proximal control elements Transcription start site Exon IntronExon Intron Poly-A signal sequence Transcription termination region Downstream

36. Figure 18.8-2 Enhancer (distal control elements) DNA Upstream Promoter Proximal control elements Transcription start site Exon IntronExon Intron Poly-A signal sequence Transcription termination region Downstream Poly-A signal Exon IntronExon Intron Transcription Cleaved 3 end of primary transcript 5 Primary RNA transcript (pre-mRNA)

37. Figure 18.8-3 Enhancer (distal control elements) DNA Upstream Promoter Proximal control elements Transcription start site Exon IntronExon Intron Poly-A signal sequence Transcription termination region Downstream Poly-A signal Exon IntronExon Intron Transcription Cleaved 3 end of primary transcript 5 Primary RNA transcript (pre-mRNA) Intron RNA RNA processing mRNA Coding segment 5 Cap 5 UTR Start codon Stop codon 3 UTR 3 Poly-A tail P PPG AAA  AAA

38. Figure 18.9 DNA Activation domain DNA-binding domain

39. Some transcription factors function as repressors, inhibiting expression of a particular gene by a variety of methods Some activators and repressors act indirectly by influencing chromatin structure to promote or silence transcription © 2011 Pearson Education, Inc.

40. Activators DNA Enhancer Distal control element Promoter Gene TATA box Figure 18.10-1

41. Activators DNA Enhancer Distal control element Promoter Gene TATA box General transcription factors DNA- bending protein Group of mediator proteins Figure 18.10-2

42. Activators DNA Enhancer Distal control element Promoter Gene TATA box General transcription factors DNA- bending protein Group of mediator proteins RNA polymerase II RNA synthesis Transcription initiation complex Figure 18.10-3

43. Mechanisms of Post-Transcriptional Regulation Transcription alone does not account for gene expression Regulatory mechanisms can operate at various stages after transcription Such mechanisms allow a cell to fine-tune gene expression rapidly in response to environmental changes © 2011 Pearson Education, Inc.

44. RNA Processing In 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 © 2011 Pearson Education, Inc. Animation: RNA Processing

45. Exons DNA Troponin T gene Primary RNA transcript RNA splicing or mRNA 1 1 11 2 2 2 2 3 3 3 4 4 4 5 5 5 5 Figure 18.13

46. 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 Nucleotide sequences that influence the lifespan of mRNA in eukaryotes reside in the untranslated region (UTR) at the 3 end of the molecule © 2011 Pearson Education, Inc. Animation: mRNA Degradation

47. Initiation of Translation The initiation of translation of selected mRNAs can be blocked by regulatory proteins that bind to sequences or structures of the mRNA –Within the untranslated region at the 5’ or 3’ end preventing attachment of ribosomes Alternatively, translation of all mRNAs in a cell may be regulated simultaneously For example, translation initiation factors are simultaneously activated in an egg following fertilization –A cytoplasic enzyme adds more adenine nucleotide prompting translation to begin © 2011 Pearson Education, Inc. Animation: Blocking Translation

48. Protein Processing and Degradation After translation, various types of protein processing, including cleavage and the addition of chemical groups, are subject to control- to yield functional protein molecules –Regulatory proteins are activated or inactivated by the reversible addition of phosphate groups –Cell surface proteins are transported to target locations in order for cells to function Selective degradation regulates the lifetime of a functional protein Proteasomes are giant protein complexes that bind protein molecules and degrade them © 2011 Pearson Education, Inc. Animation: Protein Processing Animation: Protein Degradation

49. Figure 18.14 Protein to be degraded Ubiquitin Ubiquitinated protein Proteasome Protein entering a proteasome Proteasome and ubiquitin to be recycled Protein fragments (peptides)

50. 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 © 2011 Pearson Education, Inc.

51. Types of 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 Oncogenes are cancer-causing genes Proto-oncogenes are the corresponding normal cellular genes that are responsible for normal cell growth and division –They can code for proteins associated with cell growth Conversion of a proto-oncogene to an oncogene can lead to abnormal stimulation of the cell cycle © 2011 Pearson Education, Inc.

52. Figure 18.23 Proto-oncogene DNA Translocation or transposition: gene moved to new locus, under new controls Gene amplification: multiple copies of the gene New promoter Normal growth- stimulating protein in excess Point mutation: within a control element within the gene Oncogene Normal growth- stimulating protein in excess Hyperactive or degradation- resistant protein

53. Proto-oncogenes can be 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 © 2011 Pearson Education, Inc.

54. Tumor-Suppressor Genes Tumor-suppressor genes help prevent uncontrolled cell growth –can encode proteins that promote DNA repair or cell-cell adhesion 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 © 2011 Pearson Education, Inc.

55. 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 –characteristic of the product of the p53 gene is that It is an activator for other genes (such as p21 which allow time for the cell to repair its DNA) –Thus termed the guardian angel of the genome Mutations in the p53 gene prevent suppression of the cell cycle © 2011 Pearson Education, Inc.

56. Inherited Predisposition and Other 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, and tests using DNA sequencing can detect these mutations © 2011 Pearson Education, Inc.