Chapter 15- Regulation of Gene Expression

1. Chapter 15- Regulation of Gene Expression

2. Following the Big Ideas • Energy and Homeostasis- • Timing and coordination of specific events are necessary for the normal development of an organism, and these events are regulated by a variety of mechanisms. • Evolution- • Changes in DNA can lead to phenotypic variation which can be acted upon by natural selection. • Bacterial genomes benefit from their ability to evolve rapidly.

3. Following the Big Ideas • Information and Signaling- • DNA, and in some cases RNA, is the primary source of inheritable information. • A variety of intercellular and intracellular signal transmissions mediate gene expression. • Gene regulation results in differential gene expression, leading to cell specialization. • Eukaryotes fine tune gene expression and modify gene products. • Biological systems have multiple processes that increase genetic variation. • Interactions and Systems- • Regulation of gene activity involves intricate interactions with internal and external factors. • Variation in molecular units provides cells with a wider range of functions.

4. Overview: Differential Expression of Genes Prokaryotes and eukaryotes alter gene expression in response to their changing environment Multicellular eukaryotes also develop and maintain multiple cell types Gene expression is often regulated at the transcription stage, but control at other stages is important, too See Bozeman video: https://www.youtube.com/watch?v=3S3ZOmleAj0&list=PLFCE4D99C4124A27A&index=39

5. Concept 15.1: Bacteria often respond to environmental change by regulating transcription Natural selection has favored bacteria that produce only the gene products needed by the cell A cell can regulate the production of enzymes by feedback inhibition or by gene regulation A bacterium can tune its metabolism to the changing environment and food sources This metabolic control occurs on two levels: Adjusting activity of metabolic enzymes Regulating genes that encode metabolic enzymes Gene expression in bacteria is controlled by a mechanism described as the operon model

6. Prokaryotic control of gene expression • regulation of enzyme activity- enzyme activity of many enzymes may increase or decrease in response to chemical cues • the end product of an anabolic pathway may turn off its own production by inhibiting activity of an enzyme at the beginning of the pathway (feedback inhibition) • regulation of gene expression- enzyme concentrations may rise and fall in response to cellular metabolic changes that switch genes on and off • accumulation of a product may trigger a mechanism that inhibits transcription of mRNA production by genes that code for an enzyme at the beginning of the pathway (gene repression)

7. Figure 15.2 Precursor Feedbackinhibition trpE gene Enzyme 1 Regulation of gene expression trpD gene Enzyme 2 trpC gene trpB gene Enzyme 3 trpA gene Tryptophan (a) Regulation of enzyme activity (b) Regulation of enzyme production

8. Operons: The Basic Concept • In bacteria, genes are often clustered into operons, composed of • An operator, an “on-off” switch • A promoter- RNA polymerase attaches • Genes for metabolic enzymes • An operon can be switched off by a protein called a repressor • Repressors are proteins that are coded for by a regulatory gene upstream from the operon. • A corepressor is a small molecule that cooperates with a repressor to switch an operon off • See Bozeman video: • https://www.youtube.com/watch?v=10YWgqmAEsQ

9. The Operon Model- Jacob and Monod

10. Figure 15.3a DNA trp operon Regulatory gene Promoter Promoter Genes of operon trpB trpR trpE trpD trpC trpA Operator RNA polymerase Stop codon Start codon mRNA 3 mRNA 5 5 D C B A E Protein Inactive repressor Polypeptide subunits that make up enzymes for tryptophan synthesis (a) Tryptophan absent, repressor inactive, operon on

11. Figure 15.3b DNA No RNA made mRNA Protein Active repressor Tryptophan (corepressor) (b) Tryptophan present, repressor active, operon off

12. 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 then 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

13. Repressible and Inducible Operons: Two Types of Negative Gene Regulation • 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 • The classic example of an inducible operon is the lac operon • The lac operon 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

15. For the lac operon, the inducer is allolactose, formed from lactose that enters the cell Enzymes of the lactose pathway are called inducible enzymes Analogously, the enzymes for tryptophan synthesis are said to be repressible enzymes Video: lac Repressor Model

16. Figure 15.4a Regulatory gene Promoter Operator DNA IacZ lacI No RNA made 3 mRNA RNA polymerase 5 Active repressor Protein (a) Lactose absent, repressor active, operon off

17. Figure 15.4b DNA lac operon IacI IacA IacY IacZ RNA polymerase mRNA 3 mRNA 5 5 Protein Permease -Galactosidase Transacetylase Inactive repressor Allolactose(inducer) (b) Lactose present, repressor inactive, operon on

19. 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

20. Both the repressible and inducible operon are types of NEGATIVE gene regulation because both are turned OFF by the active form of the repressor protein. In either type of operon, binding of a specific repressor protein to the operator shuts off transcription.

21. Factors Affecting Ability of Repressor to Bind to Operator Repressible Operons Co-Repressor : Activates a Repressor Seen in the trp Operon Co-Repressor is tryptophan Turns the normally “on” Operon “off” when enough tryptophan has been synthesized or absorbed. Pathway end product switches off its own production by repressing enzyme synthesis Generally function in anabolic (synthesis) metabolic pathways Inducible Operons Inducer: Inactivates a Repressor, Induces the Gene to be Transcribed Seen in the lac Operon Inducer is allolactose Turns the normally “off” Operon “on” when a specific metabolite (allolactose) inactivates the repressor protein Enzyme synthesis is switched on by the nutrient the pathway uses Generally function in catabolic (hydrolysis) metabolic pathways (Inducers and corepressors are small molecules that interact with regulatory proteins (repressors) and/or regulatory sequences (operator of the operon) Prokaryotic Gene Regulation

22. Positive Gene Regulation • When glucose, a preferred food source of E. coli is scarce, the lac operon is activated by the binding of CAP • When glucose levels increase, CAP detaches from the lac operon, slowing transcription, even if lactose is present. • Active CAP is like a volume control.

23. Positive Gene Regulation Some operons are also subject to positive control through a stimulatory activator protein, such as catabolite activator protein (CAP) which binds to the lac operon stimulating transcription. E. coli will preferentially use glucose when it is present in the environment When glucose is scarce, CAP (catabolite activator protein) acts as an activator of transcription CAP is activated by binding with cyclic AMP (cAMP)

24. Activated CAP attaches to the promoter of the lac operon and increases the affinity of RNA polymerase, thus accelerating transcription When glucose levels increase, CAP detaches from the lac operon, and transcription proceeds at a very low rate, even if lactose is present CAP helps regulate other operons that encode enzymes used in catabolic pathways

25. Figure 15.5a Promoter DNA lacI IacZ Operator CAP-binding site RNApolymerasebinds andtranscribes Active CAP cAMP Inactive lacrepressor Inactive CAP Allolactose (a) Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized

26. Figure 15.5b Promoter lacI DNA IacZ Operator CAP-binding site RNA polymerase lesslikely to bind Inactive CAP Inactive lacrepressor (b) Lactose present, glucose present (cAMP level low): little lac mRNA synthesized

27. Prokaryotic Gene Regulation Review: Structure/Function of Prokaryotic Chromosomes • shape (circular/nonlinear/loop) • less complex than eukaryotes (no histones (proteins)/less elaborate structure/folding) • size (smaller size/less genetic information/fewer genes) • replication method (single origin of replication/rolling circle replication) • transcription/translation may be coupled • generally few or no introns (noncoding segments) • majority of genome expressed • operons are used for gene regulation and control • NOTE: plasmids – more common but not unique to prokaryotes/not part of prokaryote chromosome

28. Concept 15.2: Eukaryotic gene expression is regulated at many stages 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 (cell specialization) RNA molecules play many roles in regulating gene expression in eukaryotes All organisms must regulate which genes are expressed at any given time

29. Eukaryotic DNA is: • complexed with a large amount of protein to form chromatin • highly extended during interphase • condensed into short thick discrete chromosomes during mitosis • contain enormous amounts of DNA which requires an elaborate system of DNA packing to fit all of the cell’s DNA into the nucleus

30. 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 • In each type of differentiated cell, a unique subset of genes is expressed • Abnormalities in gene expression can lead to diseases including cancer • Gene expression is regulated at many stages • Regulatory genes are sequences of DNA that code for a protein or an RNA molecule that functions to turn on, turn off, or change the rate of gene expression.

31. Figure 15.6a Signal NUCLEUS Chromatin Chromatin modification:DNA unpacking involvinghistone acetylation andDNA demethylation DNA Gene availablefor transcription Gene Transcription Exon RNA Primary transcript Intron RNA processing Tail mRNA in nucleus Cap Transport to cytoplasm CYTOPLASM

32. Figure 15.6b CYTOPLASM mRNA in cytoplasm Translation Degradation of mRNA Polypeptide Protein processing, suchas cleavage andchemical modification Active protein Degradationof protein Transport to cellulardestination Cellular function(such as enzymaticactivity, structural support)

33. Chromatin structure is based on successive levels of DNA packing • Eukaryotic DNA is precisely combined with a large amount of protein • Eukaryotic chromosomes contain an enormous amount of DNA relative to their condensed length

34. Regulation of Chromatin Structure In all organisms, a common control point for gene expression is at transcription The greater complexity of eukaryotic cell structure and function provides opportunities for regulating gene expression at many additional stages The structural organization of chromatin packs DNA into a compact form and also helps regulate gene expression in several ways The location of a gene promoter relative to nucleosomes and scaffold or lamina attachment sites can influence gene transcription

35. Genes within highly condensed heterochromatin are usually not expressed Chemical modifications to histone proteins and DNA can influence chromatin structure and gene expression

36. Histone Modifications and DNA Methylation In histone acetylation, acetyl groups are attached to positively charged lysines in histone tails This generally loosens chromatin structure, promoting the initiation of transcription The addition of methyl groups (methylation) can condense chromatin and lead to reduced transcription

37. A simple model of histone tails and the effect of histone acetylation Figure 18.7 Histone tails DNA double helix Amino acidsavailablefor chemicalmodification Nucleosome(end view) (a) Histone tails protrude outward from a nucleosome Acetylated histones Unacetylated histones (b) Acetylation of histone tails promotes loose chromatinstructure that permits transcription

38. DNA methylation is the addition of methyl groups to certain bases in DNA, usually cytosine Individual genes are usually more heavily methylated in cells where they are not expressed Once methylated, genes usually remain so through successive cell divisions After replication, enzymes methylate the correct daughter strand so that the methylation pattern is inherited

39. Epigenetic Inheritance • Although the chromatin modifications just discussed do not alter DNA sequence, they may be passed to future generations of cells • The inheritance of traits transmitted by mechanisms not directly involving the nucleotide sequence is called epigenetic inheritance • Epigenetic modifications can be reversed, unlike mutations in DNA sequence “When fruit flies are exposed to certain chemicals, at least 13 generations of their descendants are born with bristly outgrowths on their eyes. Another study found that exposing a pregnant rat to a chemical that alters reproductive hormones leads to generations of sick offspring. Yet another study shows higher rates of heart disease and diabetes in the children and grandchildren of people who were malnourished in adolescence. In these cases, the source of the variation in subsequent generations was not DNA. Rather, the new traits were carried on through epigenetic means.”University of Chicago Press Journals (2009, May 20). Epigenetics: 100 Reasons To Change The Way We Think About Genetics.

40. 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

41. 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 for the precise regulation of gene expression in different cell types

42. Figure 15.8 Poly-A signalsequence Enhancer(distal controlelements) Proximalcontrol elements Transcriptionterminationregion Transcriptionstart site Exon Intron Intron Exon Exon DNA Upstream Down-stream Promoter Transcription Poly-Asignal Intron Intron Exon Exon Primary RNAtranscript(pre-mRNA) Exon Cleaved 3end ofprimarytranscript 5 RNA processing Intron RNA Coding segment mRNA 3 G P P P AAAAAA Stopcodon Startcodon UTR 5 Cap 5 UTR Poly-Atail 3

43. 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 interaction between control elements and specific transcription factors

44. 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 Enhancers and Specific Transcription Factors

45. 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

47. Figure 15.9 Activationdomain DNA-bindingdomain DNA

48. Bound activators are brought into contact with a group of mediator proteins through DNA bending The mediator proteins in turn interact with proteins at the promoter These protein-protein interactions help to assemble and position the initiation complex on the promoter Animation: Transcription Initiation

49. Figure 15.10-3 Promoter Activators Gene DNA Distal controlelement TATA box Enhancer General transcriptionfactors DNA- bendingprotein Group of mediator proteins RNApolymerase II RNApolymerase II Transcriptioninitiation complex RNA synthesis

50. 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