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Eukaryotic Regulation

Eukaryotic Regulation. Chapter 17 Sections:17.2, 17.3 - 17.7 &17.9. Eukaryotic Regulation Differs from Prokaryotic Regulation. Eukaryotes contain much greater amounts of genetic information Many chromosomes

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Eukaryotic Regulation

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

  2. 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 Chapter 17: Eukaryotic Gene Expression

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

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

  5. Chapter 17: Eukaryotic Gene Expression

  6. Transcription Control Chapter 17: Eukaryotic Gene Expression

  7. 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 Chapter 17: Eukaryotic Gene Expression

  8. 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 Chapter 17: Eukaryotic Gene Expression

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

  10. Chapter 17: Eukaryotic Gene Expression

  11. Chapter 17: Eukaryotic Gene Expression

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

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

  14. Chapter 17: Eukaryotic Gene Expression

  15. Positive Transcription Factors(True Activators) • 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) Chapter 17: Eukaryotic Gene Expression

  16. Chapter 17: Eukaryotic Gene Expression

  17. 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 – N3 – His Chapter 17: Eukaryotic Gene Expression

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

  19. Zinc Finger Chapter 17: Eukaryotic Gene Expression

  20. Zinc Finger Chapter 17: Eukaryotic Gene Expression

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

  22. Leucine Zipper Chapter 17: Eukaryotic Gene Expression

  23. Transcription Control Chapter 17: Eukaryotic Gene Expression

  24. 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 Chapter 17: Eukaryotic Gene Expression

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

  26. 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 Chapter 17: Eukaryotic Gene Expression

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

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

  29. GAL Genes Chapter 17: Eukaryotic Gene Expression

  30. 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 Chapter 17: Eukaryotic Gene Expression

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

  32. 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 Chapter 17: Eukaryotic Gene Expression

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

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

  35. 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 Chapter 17: Eukaryotic Gene Expression

  36. 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 Chapter 17: Eukaryotic Gene Expression

  37. 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 Chapter 17: Eukaryotic Gene Expression

  38. 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 Chapter 17: Eukaryotic Gene Expression

  39. 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 Chapter 17: Eukaryotic Gene Expression

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

  41. 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. Chapter 17: Eukaryotic Gene Expression

  42. 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. Chapter 17: Eukaryotic Gene Expression

  43. 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. Chapter 17: Eukaryotic Gene Expression

  44. 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 Chapter 17: Eukaryotic Gene Expression

  45. 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) Chapter 17: Eukaryotic Gene Expression

  46. 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 Chapter 17: Eukaryotic Gene Expression

  47. 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 Chapter 17: Eukaryotic Gene Expression

  48. 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 Chapter 17: Eukaryotic Gene Expression

  49. 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 Chapter 17: Eukaryotic Gene Expression

  50. 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 Chapter 17: Eukaryotic Gene Expression

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