1. Chapter 17: Eukaryotic Gene Expression1 Eukaryotic Regulation Chapter 17 Sections:17.2, 17.3 - 17.7 &17.9

2. Chapter 17: Eukaryotic Gene Expression2 Eukaryotic Regulation Differs from Prokaryotic Regulation  Eukaryotes contain much greater amounts of genetic information  Many chromosomes  Genetic information is segregated from nucleus to cytoplasm; Prokaryotes use cytoplasm only  Posttranscriptional Regulation  Eukaryotic mRNA has longer half-life  Eukaryotic mRNA is more stable

3. Chapter 17: Eukaryotic Gene Expression3 Types of Gene Regulation  Control of Gene Expression Chromosomal Organization  Chromatin Remodeling Transcription  Promoters  Enhancers (enhanceosome)  Upstream Activating Sequences (UAS)  Transcription Initiation Complex  Activators

4. Chapter 17: Eukaryotic Gene Expression4 Control of Gene Expression (continued)  mRNA Degradation Translational Control  RNA Silencing RNAi  mRNA Processing Alternative splicing

5. Chapter 17: Eukaryotic Gene Expression5

6. 6 Transcription Control

7. Chapter 17: Eukaryotic Gene Expression7 Transcriptional Control  Why do you need a promoter? Recognition site for binding of RNA polymerase Necessary for initiation of transcription Upstream from gene start site Several hundred nucleotides in length

8. Chapter 17: Eukaryotic Gene Expression8 Transcriptional Control  Actual Promoter : TATA BOX (-25 to –35)  Sequences within the promoter region that function as enhancers are: 1. CAAT or CCAAT (cat box) -70 to –80 2. GGGCGG (GC box) -110

9. Chapter 17: Eukaryotic Gene Expression9 Initiation Complex for Transcription 1. TFIID has 2 subunits : TBP and TAF 2. First, TBP subunit binds to TATA box 3. TAF promotes a conformational change in the DNA which allows other TF to bind (commitment stage) 4. Pol II leaves TATA box and transcribes (promoter clearance)

10. Chapter 17: Eukaryotic Gene Expression10

11. Chapter 17: Eukaryotic Gene Expression11

12. Chapter 17: Eukaryotic Gene Expression12 Enhancers 1. Necessary for full level of transcription 2. Responsible for tissue-specific gene expression 3. Able to bind transcription factors by associating with RNA polymerase forming DNA loops

13. Chapter 17: Eukaryotic Gene Expression13 Enhancers Different from Promoters because: 1. No fixed position – upstream, downstream or within gene 2. Different orientation 3. Affect transcription of other genes if moved to another location

14. Chapter 17: Eukaryotic Gene Expression14

15. Chapter 17: Eukaryotic Gene Expression15 Positive Transcription Factors (True Activators) A. Proteins with at least two functional domains B. Functional Domains: 1. Bind to the enhancer (DNA binding domain) 2. Protein-Protein interaction with RNA Pol or other transcription factors (trans- activating domain)

16. Chapter 17: Eukaryotic Gene Expression16

17. Chapter 17: Eukaryotic Gene Expression17 Positive Transcription Factors (True Activators) DNA Binding Domains 1. Helix-Turn-Helix (homeodomain) – 180 kb or 60 amino acids/ bind to major and minor grooves as well as backbone 2. Zinc Fingers – Cys and His covalently bind zinc atom/bind major and minor goove Cys – N 2-4 - Cys – N 12-14 –His – N 3 – His

18. Chapter 17: Eukaryotic Gene Expression18 Helix-Turn-Helix

19. Chapter 17: Eukaryotic Gene Expression19 Zinc Finger

20. Chapter 17: Eukaryotic Gene Expression20 Zinc Finger

21. Chapter 17: Eukaryotic Gene Expression21 Positive Transcription Factors (True Activators) 3. Leucine Zipper – 4 leucine residues spaced 7 amino acids apart and flanked by basic amino acids - leucine regions form  -helix - leucine regions dimerize and and zip together

22. Chapter 17: Eukaryotic Gene Expression22 Leucine Zipper

23. Chapter 17: Eukaryotic Gene Expression23 Transcription Control

24. Chapter 17: Eukaryotic Gene Expression24 Transcription Control: GAL genes  Galactose-utilizing genes  Part of metabolic pathway to metabolize galactose in yeast  Follow the activation of genes GAL 1, 7, 10 that are located near one another on the DNA  Genes are made in response to the presence of galactose  Gal4p and Gal80p are regulatory proteins in the process and UAS-G is the DNA sequence

25. Chapter 17: Eukaryotic Gene Expression25 Transcription Control: GAL genes

26. Chapter 17: Eukaryotic Gene Expression26 Transcription Control: GAL genes  In the absence of galactose, GAL 80p is bound to GAL 4p and GAL 4p is bound to the regulatory DNA sequence (UAS-G) Under these conditions, transcription of GAL 1, 7, 10 is inhibited  In the presence of galactose, a metabolite of galactose binds to GAL 80p  GAL 4p is then phosphorylated initiating a change in conformation  GAL 4p is now capable of activating transcription

27. Chapter 17: Eukaryotic Gene Expression27 Control of GAL Genes

28. Chapter 17: Eukaryotic Gene Expression28 Transcription Control: GAL genes Fig. 17.5

29. Chapter 17: Eukaryotic Gene Expression29 GAL Genes

30. Chapter 17: Eukaryotic Gene Expression30 Transcription Control: Steroid Hormone  Not many changes in the external environment of cell in an animal  Hormones are secreted by cells in the animal and can signal changes from the environment  Peptide hormones bind to extra cellular receptors and steroid hormones bind to intracellular receptors

31. Chapter 17: Eukaryotic Gene Expression31 Transcription Control: Steroid Hormone

32. Chapter 17: Eukaryotic Gene Expression32 Transcription Control: Steroid Hormone  Steroid hormones often bind to cytoplasmic receptor and translocated to the nucleus where the complex acts  In the nucleus the complex binds to the DNA at a specific sequence  Hormones are potent regulators of gene expression, but only affect cells that produce the receptor that the particular hormone binds

33. Chapter 17: Eukaryotic Gene Expression33 Transcription Control: Steroid Hormone

34. Chapter 17: Eukaryotic Gene Expression34 Transcription Control: Steroid Hormone

35. Chapter 17: Eukaryotic Gene Expression35 Transcription Control: Steroid Hormone  Steroid hormone control of gene expression Important in development and physiological regulation Because receptor is needed, have tissue or cell type specific effects Specific for certain hormone receptor Usually found in a small number of cells Can affect tc, mRNA stability, mRNA processing

36. Chapter 17: Eukaryotic Gene Expression36 Transcription Control: Steroid Hormone  Steroid hormone control of gene expression No hormone then the receptor is inactive and bound to a chaperone protein Steroid hormone enters cell and binds to its specific receptor Chaperone is displaced Hormone binds receptor = activation Complex is transported and acts in the nucleus

37. Chapter 17: Eukaryotic Gene Expression37 Transcription Control: Steroid Hormone  Steroid hormone control of gene expression Hormone-receptor complex binds to specific DNA binding element  Transcription activation or repression depending on the complex Complex binds to the steroid hormone response element (HRE) in the DNA HRE’s are in the enhancer region and in multiple copies

38. Chapter 17: Eukaryotic Gene Expression38 Transcription Control  Transcription of a gene is also affected by the proteins bound to the DNA (histones)  DNA is less compacted in regions where DNA is transcribed  Nucleosomes are not removed  Generally physically inhibit gene transcription  Transcription can occur in the presence of nucleosomes when they are chemically modified  DNA Methylation – CpG islands/X chromosome

39. Chapter 17: Eukaryotic Gene Expression39 Control of mRNA  mRNA processing—regulation of production of mature mRNA Alternative poly-A sites Alternative/differential splicing CALC gene employs both in different cell types

40. Chapter 17: Eukaryotic Gene Expression40 Control of mRNA Fig. 17.7

41. Chapter 17: Eukaryotic Gene Expression41 Control of mRNA  Evaluate gene expression of the human calcitonin gene (CALC) in thyroid cells and neurons.  Thyroid cells Poly(A) signal after exon 4 is used Removed introns 1-4 and join exons 1-4 to make calcitonin mRNA is translated.

42. Chapter 17: Eukaryotic Gene Expression42 Control of mRNA  Evaluate gene expression of the human calcitonin gene (CALC) in thyroid cells and neurons.  Neurons Poly(A) signal after exon 5 is used Remove all introns and exon 4 is removed as well; join exons 1, 2, 3, 5 to make CGRP mRNA mRNA is translated.

43. Chapter 17: Eukaryotic Gene Expression43 Posttranslational modification  Evaluate gene expression of the human calcitonin gene (CALC) in thyroid cells and neurons. In both cell types the mRNA is translated into a protein that needs processing—pre-hormone or pre-protein This allows the protein to be synthesized and be present in the cell, but NOT be active.

44. Chapter 17: Eukaryotic Gene Expression44 Posttranslational modification When the proteins are needed, a protease cleaves the pre-portion of the protein and the remainder of the polypeptide becomes active  Calcitonin is produced in thyroid cells—hormone that helps the kidney to retain calcium; Exon 4 encodes the active protein  cGRP is produced in neurons—found in hypothalamus and has neuromodulary/growth promoting properties; Exon 5 encodes the active protein

45. Chapter 17: Eukaryotic Gene Expression45 Control of Translation  Shortened poly(A) tails prevent translation Poly(A) tails are needed for translation initiation mRNAs that are ‘stored’ and prevented from being translated have short Poly(A) tails (15-90 A’s long)

46. Chapter 17: Eukaryotic Gene Expression46 Control of Translation  Shortened poly(A) tails prevent translation Tails may be trimmed (deadenylation enzymes) or they may be short at synthesis. Deadenylation enzymes recognize AU rich element (ARE) in the 3’ UTR of the mRNA and remove A’s from the tail Other enzymes may recognize ARE in the 3’ UTR and lengthen the poly(A) tail when it is time to translate the mRNA

47. Chapter 17: Eukaryotic Gene Expression47 Control of mRNA  mRNA stability—how long the mRNA is found in the cell (RNA turnover) The longer the mRNA is found in the cell, the more copies of protein are made. Stability of mRNA varies greatly from gene to gene Important way to control gene expression

48. Chapter 17: Eukaryotic Gene Expression48 Control of mRNA  mRNA stability—how long the mRNA is found in the cell (RNA turnover) Stability can be controlled by molecules present in the cell Signals found in the 5’ or 3’ UTR Control when the mRNA is degraded

49. Chapter 17: Eukaryotic Gene Expression49 Control of mRNA  mRNA stability—how long the mRNA is found in the cell (RNA turnover) 2 major pathways  Deadenylation –dependent decay pathway  Deadenylation-independent decay pathway

50. Chapter 17: Eukaryotic Gene Expression50 Control by Protein Degradation  Posttranslational control  Controls how long the protein is present and active in the cell  Controlled by attachment of the protein ubiquitin to the protein being targeted for degradation  Signals for the protein to be degraded by the proteasome  N-terminus of the protein will determine its stability by determining the rate that ubiquitin can bind to the protein