Chapter 7: Control of Gene Expression

1. Chapter 7: Control of Gene Expression

2. Control of Gene Expression • Different cell types differ dramatically in structure and function • same genome • Cell differentiation depends on gene expression

3. Control of Gene Expression Evidence for preservation of genome during cell differentiation

4. Control of Gene Expression • Different Cell Types Synthesize Different Sets of Proteins • How many differences are there btwn any one cell type and another • Many processes are common to all cells • Some processes are cell specific • Cell expresses ~10,000-20,000 of its 30,000 genes; level of expression of almost every gene varies from cell to cell

5. Control of gene Expression Cells Can Change Expression of its Genes in Response to External Signals • Different cell types respond in different ways to same extracellular signal= general feature of cell specialization • Example: Liver and adipocyte cells respond differently to glucocorticoid Liver Cell Tyrosine aminotransferase Adipocyte Tyrosine aminotransferase

6. Control of Gene Expression For most genes transcription control is most important

7. Control of Gene Expression 2 Fundamental Components to Transcriptional Gene Regulation 1. Gene Regulatory Proteins 2. Short Stretches of DNA of Defined Sequence

8. Control of Gene Expression • Outside of DNA Helix Read by Proteins • GRP recognizes specific nucleotide sequence • Information in form of: H-bond acceptors H-bond donors Hydrophobic patches • Bind to Major Groove

9. Control of Gene Expression GRPs bind to major groove where patterns for ea of four base-pair arrangements are distinct

10. Control of Gene Expression Geometry of Double Helix Depends on Nucleotide Sequence • Some nucleotide sequences cause DNA to bend • AAAANNN • If repeated every 10 bp DNA appears unusually curved

11. Control of Gene Expression DNA must be flexible for binding of GRPs

12. Control of Gene Expresion Short DNA Sequences Fundamental Components of Genetic Switches • GRP recognition sequence generally < 20 bp • Thousands of such DNA sequences identified ea of which is recognized by different GRP

13. Control of Gene Expression GRP – DNA Interactions • Exact fit btwn DNA and protein • H-bonds, ionic bonds, hydrophobic • > 20 contacts • Tight and specific

14. Control of Gene Expression Major Structural Motifs of GRPs • Helix-turn-helix • Homeodomain • Zinc Finger • Leucine Zipper • Helix-Loop-Helix

15. Control of Gene Expression Helix-Turn-Helix • Most common • C-terminal helix= recognition helix • aa in recognition helix define specificity • Structure of GRP varies outside HTH; HTH presented in unique way

16. Control of Gene Expression Homeodomain • Special type of helix-turn-helix • Conserved stretch of 60 aa • HTH motif always surrounded by same structure- homeodomain • Master regulators of development

17. Control of Gene Expression Zinc Finger Proteins • α helix and β sheet • (2) α helices

18. Control of Gene Expression Leucine Zipper • Clothespin • Helices held together by short coiled coil region of hydrophobic residues often leucines

19. Control of Gene Expression Helix-Loop-Helix • Short α helix connected to another via loop • Flexible loop for packing

20. Control of Gene Expression Heterodimerization • Enhances the repertoire of DNA binding specificities • Combinatorial control

21. Control of Gene Expression Is it possible to predict DNA sequence to which GRP’s bind?

22. Control of Gene Expression Gel Mobility Shift Assay to Detect GRPs • effect of a bound protein on the migration of DNA in an electric field

23. Control of Gene Expression DNA Affinity Chromatography to Purify GRPs Purification of GRP > 10,000X

24. Control of Gene Expression How do we determine the sequence to which a particular GRP binds?

25. Control of Gene Expression • Chromatin Immunoprecipitation • Identifies sequences occupied by GRPs in living cells • Used to identify direct targets of GRPs

26. How Genetic Switches Work Tryptophan Operon Operon= a cluster of genes transcribed as a single mRNA Operator = short region of DNA in bact. that controls transcription of an adjacent gene

27. How Genetic Switches Work Tryptophan Repressor = a Simple On/Off Switch

28. How Genetic Switches Work Repressor= protein binds to DNA to prevent transcription of adjacent gene Activator = protein that binds to DNA and promotes the transcription of adjacent gene

29. How Genetic Switches Work • CAP= Catabolite Activator Protein • Promotes transcription of genes that enable E. coli to use alternative carbon sources when glucose is not available • glucose cAMP • cAMP binds to CAP enabling CAP to bind to sequences near target promoters to promote transcription

30. How Genetic Switches Work More complicated genetic switches combine positive and negative controls Lac Operon- under the control of transcriptional activator and transcriptional repressor

31. How Genetic Switches Work Regulation of Transcription in Eukaryotes is More Complex • GRPs can act even when positioned 1000’s bp away from promoter • RNA Pol II cannot initiate transcription on its own, requires GTFs • Packaging of DNA in chromain

32. How Genetic Switches Work • Eucaryotic Gene Control Region • Promoter and all regulatory sequences to which GRPs bind to control transcription • > 50,000 bp, not unusual • Packaged in nucleosomes and higher order forms of chromatin

33. How Genetic Switches Work • Eucaryotic GRPs • 5-10% of human genome • Vary from one control region to next • Present in sm amts, <0.01% total protein • Most recognize specific DNA sequences; others assemble on other DNA bound proteins • Allow genes to be turned on and off very specifically

34. How Genetic Switches Work Eucaryotic Gene Activator Proteins Promote Assembly of RNA Polymerase and GTFs at Transcription Start • Gene Activator Proteins have Modular Design: • DNA Binding Domain • Activator Domain

35. How Genetic Switches Work • Mechanism of Gene Activator Proteins Varied but All Promote Assembly of GTFs and RNA Pol • Interact w/ initiation complex to recruit RNA Pol • Interact directly w/RNA Pol and GTFs • Change chromatin structure around promoter

36. How Genetic Switches Work GRPs can affect: • prescribed ordered assembly of GTFs and RNA Polymerase • Recruitment of RNA Polymerase holoenzyme to promoter

37. How Genetic Switches Work • Gene Activator Proteins Promote Assembly of GTFs and RNA Pol By • Modification of Local Chromatin Structure Recruiting histone acetyl transferases histone remodeling complexes

38. How Genetic Switches Work Gene Activator Proteins Work Synergistically

39. How Genetic Switches Work EX: Complexity of How Gene Activator Proteins May Ultimately Increase Transcription Rate

40. How Genetic Switches Work Eucaryotic Repressors Inhibit Transcription in Variety of Ways

41. How Genetic Switches Work • Eucaryotic GRPs and Combinatorial Control • Function as unit to generate complexes whose function depends on final assembly of all components • Not designated activators or repressors • DNA acts as nucleation site for assembly • Can participate in > one type of reg. complex • Coactivators and corepressors • enhancesome

42. How Genetic Switches Work • Eve-skipped gene is a complex multicomponent genetic switch in drosophilia • Drosophilia development • Eve expressed when embryo single giant multinucleated cell • Cytoplasm=mixture of GRPs distributed unevenly along length of embryo • Nuclei originally identical but later express diff genes cuz exposed to diff GRPs

43. How Genetic Switches Work • Eve Expression • Regulatory sequence reads conc of GRPs at ea position along length of embryo • Expressed in 7 stripes 5-6 nuclei wide precisely positioned along anterior- posterior axis

44. How Genetic Switches Work • Regulatory Region of Eve Gene • ~20,000 bp binds >20 proteins • Series of regulatory modules • Regulatory modules contain multiple reg sequences responsible for specifying a particular stripe

45. How Genetic Switches Work • Expression of Stripe 2 • Dictated by 2 gene activator proteins and 2 gene repressor proteins • Transcription occurs when activators Biocoid and Hunchback are high and repressors Kruppel and Giant are low

46. How Genetic Switches Work Combinatorial Control • Heterodimerization of GRPs in soln • Assembly of combos of GRPs into sm complexes on DNA • Many sets of grps bound simultaneous to effect transcription

47. How Genetic Switches Work Simple regulatory modules= theme of complex gene regulatory control regions in mammals • 5-10% coding capacity of mam genome= GRPs • Ea gene regulated by set of GRPs • Ea protein is product of gene that is in turn regulated by set of other proteins • Activity of GRPs regulated

48. How Genetic Switches Work Regulation of Activity of GRPs

49. How Genetic Switches Work • Human β-globin Gene • Complex regulation- 2 step process • Expressed only in RBC at specific time during development • Possesses own set of GRPs but also under control of LCR • Cells where no globin gene expressed gene cluster tightly pkged • Higher order pkging decondensed in RBS

50. How Genetic Switches Work LCR= regulatory seq that govern accessibility and expression of distant genes or gene clusters • β-thalassemia= deletion in β-globin LCR causing gene to remain transcriptionally silent– • Many LCRs present in human genome