Chapter 15: Regulation of Gene Expression

1. Chapter 15: Regulation of Gene Expression Anablepanablepwith its “4 eyes”. Upper half of lids look aerially while the lower half looks into the water. Cells of the two parts of the eye exhibit differential gene expression

2. Individual bacteria respond to environmental change by regulating their gene expression • Express only genes whose products may be needed presently by the cell. (Don’t want to waste energy) • E. colilives in the human colon & can fine-tune its metabolism to the changing environment and food sources • If tryptohan is lacking, the cell responds by activating a gene that starts a metabolic pathway to produce tryptophanfrom another food source that is present. • If tryptophan is plentiful, the bacteria stop making tryptophan to conserve energy.

3. (a) Regulation of enzyme activity (b) Regulation of enzyme production Feedback inhibition Precursor Enzyme 1 Gene 1 Regulation of gene expression Gene 2 Enzyme 2 Gene 3 Enzyme 3 – Enzyme 4 Gene 4 – Enzyme 5 Gene 5 Tryptophan Figure 18.20a, b • This metabolic control occurs on two levels • Adjusting the activity of enzymes already present. Sensitivity to chemical clues When there is a build up of tryptophan, it shuts off the production of Enzyme 1 Short term response • Regulating the genes encoding the metabolic enzymes • Tryptophan can shut down the transcription of a gene is a longer term response

4. Operons: The Basic Concept • In bacteria, genes are often clustered into groups of genes (such as the 5 for tryptophan) called operons, composed of: • An operator, an “on-off”switchwhich is a segment of DNA that lies betweenpromoter and enzyme-coding genes • A promoter – 1 for the entire operon • Genes for metabolic enzymes Animation

6. trp operon Promoter Promoter Genes of operon RNA polymerase Start codon Stop codon trpR trpD trpC trpB trpE trpA DNA Operator Regulatory gene 3 mRNA 5 mRNA 5 C E D B A Polypeptides that make up enzymes for tryptophan synthesis Inactiverepressor Protein • Tryptophan absent, repressor inactive, operonon. • RNA polymerase attaches to the DNA at the promoter and transcribes the operon’s genes. Figure 18.21a Animation • The Trpoperon • Is usually turned “On” (with no repressor protein stopping transcription) • Can be switched off by a protein called a repressor protein • trpR is a regulatory gene which is continually expressed in small amounts. It produces the repressor protein that binds to Operator to prevent transcription

7. DNA No RNA made mRNA Active repressor Protein Tryptophan (corepressor) Tryptophan present, repressor active, operonoff. As tryptophan accumulates, it inhibits its own production by activating the repressor protein. Tryptophan as a acorepressor (b) Operator trpR Regulatory gene RNA polymerase Inactiverepressor

8. Repressible and Inducible Operons: Two Types of Negative Gene Regulation • In a repressible operon • Transcription is usually on but can be repressed when small amount of Tryptophan binds toallostericallyto regulatory protein • Binding of a specific repressor protein to the operator shuts off transcription • Trpoperon • In an inducible operon • Induced by a chemical signal (Allolactose in lac operon) • Binding of an inducer to an innately inactive repressor inactivates the repressor and turns on transcription

9. Promoter Regulatorygene Operator DNA lacl lacZ NoRNAmade 3 RNApolymerase mRNA 5 Activerepressor Protein Lactose absent, repressor active, operon off. The lac repressor is innately active, and in the absence of lactose it switches off the operon by binding to the operator. (a) Figure 18.22a • The lac operon: regulated synthesis of Inducible enzymes. • Regulates the catabolism of lactose to galactose and glucose • Needs an inducer to remove repressor and start transcription • animation

10. lac operon DNA lacl lacz lacY lacA RNApolymerase 3 mRNA 5 mRNA 5' mRNA 5 -Galactosidase Permease Transacetylase Protein Inactiverepressor Allolactose(inducer) When Lactose is present, the repressor is inactive, operonis on & β –galactosidase is produced. Allolactose, an isomer of lactose, acts as the inducer by derepressesthe operon by inactivating the repressor. In this way, the enzymes for lactose utilization are induced. (b)

11. No lactose present, no need to produce enzymes to hydrolyze lactose to glucose & galactose If Lactose is present, operon needs to be turned on. Allolactose (inducer) binds to repressor, changes its shape making it drop off leaving RNA polymerase to start transcription

12. Inducible enzymes • Usually function in catabolic pathways • Lactose broken down into glucose and galactose • lacoperon to produce B - galactosidase • Repressible enzymes • Usually function in anabolic pathways • Tryptophan being produced • trpoperon to produce tryptophan

13. Regulation of both the trp and lac operons • Involves the negative control of genes, because the operons are switched off by the active form of the repressor protein

14. Positive Gene Regulation • If Lactose is present and glucose is lacking, E. coli will break down lactose for energy but how does it sense that glucose is lacking? • It senses this through an allosteric regulatory protein called CataboliteActivator Protein (CAP) that binds to a small molecule called cyclic AMP (cAMP) • CAP acts as an activator to bind to DNA to stimulate transcription of a gene (such as the lacoperon) • Think of it like cAMP pushing CAP to make RNA polymerase go faster to make the enzymes to break lactose down to glucose

15. Operator RNA polymerase can bindand transcribe Promoter DNA lacl lacZ CAP-binding site cAMP ActiveCAP Inactive lac repressor InactiveCAP (a) Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized.If glucose is scarce, the high level of cAMP activates CAP, and the lacoperon produces large amounts of mRNA for the lactose pathway. • In E. coli, when glucose, (a preferred food source), is scarce, cAMP (cyclic AMP) builds up • the lac operon is activated by the binding of a regulatory protein, catabolite activator protein (CAP) activated by the binding of cAMP to CAP changing its conformation. Thus allowing transcription • cAMP is the green light that tells CAP to floor it!

17. Promoter Operator DNA lacl lacZ CAP-binding site RNA polymerase can’t bind InactiveCAP Inactive lac repressor Lactose present, glucose present (cAMP level low): little lac mRNA synthesized.When glucose is present, cAMP is scarce, and CAPis unable to stimulate transcription. (b) Figure 18.23b • When glucose levels in an E. coli cell increases(& lactose levels are low) there is a decrease in cAMP. CAPdetaches from the lacoperon due to the release of cAMP to CAP changing its conformation, & turning it off • Animation

18. Eukaryotic gene expression is regulated at many stages • All organisms must regulate which genes are expressed at any given time. Which ones are turned on or off. • In multicellular organisms regulation of gene expression is essential for cell specialization • ~20% of protein coding genes are being expressed at any time in human cells

19. Differential Gene Expression • Almost all the cells in an organism are genetically identical with the exception of gametes • Differences between cell types result from differential gene expression, the expression of different genes by cells with the same genome • RBC from an epithelial cell to a nerve cells • Abnormalities in gene expression can lead to diseases including cancer • Gene expression is regulated at many stages

20. Signal NUCLEUS Stages in gene expression that can be regulated in eukaryotic cells. 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 mRNA in cytoplasm Translation Degradationof mRNA Polypeptide Protein processing, suchas cleavage and chemical modification Active protein Degradationof protein Transport to cellulardestination Cellular function (suchas enzymatic activity,structural support)

21. 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

22. CYTOPLASM Figure 18.6b mRNA in cytoplasm Translation Degradationof mRNA Polypeptide Protein processing, suchas cleavage and chemical modification Active protein Degradationof protein Transport to cellulardestination Cellular function (suchas enzymatic activity,structural support)

23. Regulation of Chromatin Structure • The genes within highly packed heterochromatin (DNA + proteins) are usually not expressed • Euchromatin is found in eukaryotes. Less compacted DNA • Chemical modifications to histonesand DNA of chromatin influence both chromatin structure and gene expression • Acetylation- allows transcription • Methylation – blocks transcription • Phosphorylation – promotes transcription

24. Histone Modifications For the Transcription of a gene on DNA to occur, histones must be modified to uncoil the DNA to allow for transcription • In histoneacetylation, acetyl groups (-COCH3) are attached to positively charged lysinesin histone tails • Histone tails no longer will bind to adjacent nucleosomes • This loosens chromatin structure, thereby promotes the initiation of transcription • Histoneacetylation enzymes may promote the initiation of transcription. Kosigrin animation Mc Graw Hill

25. 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

26. The addition of methyl groups – CH3 to Cystosine(methylation) to histone tails promotes condensing of chromatin. • So this turns off genes • The addition of phosphate groups (phosphorylation) next to a methylated amino acid canloosen chromatin • Turns on genes

27. DNA Methylation • DNA methylation, the addition of methyl groups to certain bases in DNA, is associated with reduced transcriptionin some species • DNA methylation can cause long-term inactivation of genes in cellular differentiation • Such as in X inactivation when the X chromosomes become methylated • Unexpressed genes are more heavily methylated than active genes.

28. Epigenetic Inheritance • Although the chromatin modifications 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 • Caused by DNA methylation and therefore certain enzymes are not produced which may be necessary for cell function • May be the cause of some cancers • Ghosts in Your Genes • Epigenetics video

30. Control & Organization of a Typical Eukaryotic Gene • 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 • Associated with most eukaryotic genes are multiplecontrol elements, segments of noncoding DNA that serve as binding sites for transcription factorsthat help regulate transcription • Control elements and the transcription factors they bind are critical to the precise regulation of gene expression in different cell types

31. A eukaryotic gene and its transcript. Proximalcontrolelements Enhancer(distal controlelements) Poly-Asignalsequence Transcriptionterminationregion Transcriptionstart site Exon Exon Intron Intron Exon DNA Upstream Downstream Promoter

32. A eukaryotic gene and its transcript. Enhancer(distal controlelements) Poly-Asignalsequence Proximalcontrolelements Transcriptionterminationregion Transcriptionstart site Exon Intron Intron Exon Exon DNA Upstream Downstream Promoter Poly-Asignal Transcription Exon Intron Intron Exon Exon Primary RNAtranscript(pre-mRNA) Cleaved3 end ofprimarytranscript 5

33. A eukaryotic gene and its transcript. Enhancer(distal controlelements) Proximalcontrolelements Poly-Asignalsequence Transcriptionterminationregion Transcriptionstart site Exon Intron Intron Exon Exon DNA Upstream Downstream Promoter Poly-Asignal Transcription Exon Intron Intron Exon Exon Primary RNAtranscript(pre-mRNA) Cleaved3 end ofprimarytranscript 5 RNA processing Intron RNA Coding segment mRNA 3 G P P P AAA AAA Startcodon Stopcodon Poly-Atail 5 UTR 5 Cap 3 UTR

34. 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

35. Enhancers and Specific Transcription Factors • 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 • These control elements could be thousands of base pairs away from the gene on the DNA strand

36. An activator is a protein that binds to an enhancer on the control factor and stimulates transcription of a gene • Activators have two domains, one that binds to DNA and a second that activates transcription • Bound activators facilitate a sequence of protein-protein interactions that result in transcription of a given gene McGraw Hill animation

37. Some transcription factors function as repressors, inhibiting expression of a particular geneby a variety of methods • Some activators and repressors act indirectly by influencing chromatin structure to promote or silence transcription

38. Promoter Activators Gene DNA Distal controlelement Enhancer TATA box A model for the action of enhancers and transcription activators

39. Promoter Activators Gene DNA Distal controlelement TATA box Enhancer Generaltranscriptionfactors DNA-bendingprotein Group of mediator proteins A model for the action of enhancers and transcription activators

40. Promoter Activators Gene DNA Distal controlelement TATA box Enhancer Generaltranscriptionfactors DNA-bendingprotein Group of mediator proteins RNApolymerase II Animation RNApolymerase II Transcriptioninitiation complex RNA synthesis A model for the action of enhancers and transcription activators

41. Enhancer Promoter Controlelements Albumin gene Cell type–specific transcription Crystallingene LENS CELLNUCLEUS LIVER CELLNUCLEUS Availableactivators Availableactivators Albumin genenot expressed Albumin geneexpressed Crystallin genenot expressed Crystallin geneexpressed (a) Liver cell (b) Lens cell

42. Enhancer Promoter LIVER CELLNUCLEUS Controlelements Albumin gene Availableactivators Crystallingene Albumin geneexpressed Crystallin genenot expressed (a) Liver cell Cell type–specific transcription

43. Enhancer Promoter LENS CELLNUCLEUS Controlelements Albumin gene Availableactivators Crystallingene Albumin genenot expressed Crystallin geneexpressed (b) Lens cell Cell type–specific transcription

44. Coordinately Controlled Genes in Eukaryotes • Unlike the genes of a prokaryotic operon, each of the co-expressed 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

45. 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

46. 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

47. Exons DNA 4 1 3 5 2 Troponin T gene PrimaryRNAtranscript 3 5 2 4 1 RNA splicing or mRNA 3 5 5 2 2 4 1 1

48. 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 • 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

49. Protein Processing and Degradation • After translation, various types of protein processing, including cleavage (such as with pro-insulin which needs to be altered to become active insulin) and the addition of chemical groups, are subject to control (phosporylation) • Chemical modifications by the addition/removal of phosphate groups. • Functional length is regulator so that the protein is only present while it is needed (such as with cyclin). • To mark a protein for destruction, ubiquitin is added to it.

50. Chromatin modification Transcription • Regulation of transcription initiation:DNA control elements in enhancers bindspecific transcription factors. • Genes in highly compactedchromatin are generally nottranscribed. • Histoneacetylation seemsto loosen chromatin structure,enhancing transcription. Bending of the DNA enables activators tocontact proteins at the promoter, initiatingtranscription. • DNA methylation generallyreduces transcription. • Coordinate regulation: Enhancer forlens-specific genes Enhancer forliver-specific genes Chromatin modification Transcription RNA processing • Alternative RNA splicing: RNA processing Primary RNAtranscript Translation mRNAdegradation mRNA or Protein processingand degradation