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Regulation of Gene Expression

Regulation of Gene Expression. David Shiuan Department of Life Science Institute of Biotechnology Interdisciplinary Program of Bioinformatics National Dong Hwa University.

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Regulation of Gene Expression

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  1. Regulation of Gene Expression David Shiuan Department of Life Science Institute of Biotechnology Interdisciplinary Program of Bioinformatics National Dong Hwa University

  2. The fundamental problem of chemical physiology and of embryology --- is to understand why tissue cells do not all express, all the time, all the potentialities inherent in their genome.Francois Jacob and Jacques MonodJ. Mol. Biol. 1961

  3. 1. Principle of gene regulation • 2. Regulation of gene expression in prokaryotes • 3. Regulation of gene expression in eukaryotes

  4. Seven processes affect the steady-state concentration of a protein

  5. Potential Points of Regulation • Synthesis of primary RNA transcript (transcription) • Posttranscriptional modification of mRNA • mRNA degradation • Protein synthesis (translation) • Posttranslational modification of proteins • Protein targeting and transport • Protein degradation

  6. Principle of gene regulation • Molecular circuits ------------------------------- • House keeping genes; constitutive gene expression • Inducible; induction; repressible; repression • RNA polymerase binds to DNA at promoters

  7. Consensus sequence for promoters that regulate expression of the E. coli heat shock genes

  8. Many prokaryotic genes are clustered and regulated in operons

  9. Lactose metabolism in E. coli

  10. They published a paper - Coordinated regulation of lac operon, Proc. French Acad. Sci. (1960)

  11. The lac operon

  12. Lac repressor binds to operator O2 and O3

  13. Lac repressor binds to operator (PDB-1BLG)

  14. Lac repressor binds to operator - Conformational change in the repressor caused by DNA binding

  15. Lac inducer IPTG, structurally similar to lactose

  16. Groups in DNA available for protein binding Shown in red- groups Can recognize proteins

  17. Protein-DNA interactions

  18. Relationship between the lac operator sequence O1 and the lac promoter

  19. DNA Binding Domain of Lac Repressor - Helix-turn-helix

  20. Surface rendering of the DNA-binding domain gray - lac repressor; blue - DNA

  21. The DNA-binding domain, but separated

  22. The zinc-finger– each Zn2+ coordinates with 2 His and 2 Cys residues

  23. Homeodomain - approx. 60 aa Homeotic genes (genes that regulate the development of body patterns) DNA-Binding Domain - helix-turn-helix

  24. Studying DNA-Protein Interactions • EMSA (electrophoretic mobility shift assay); or gel retardation assay • DNaseI footprinting experiment • DNA affinity chromatography • SPR (Surface Plasmon Resonance)/BIACORE • CD/ORD; Spefctrofluorometry; NMR

  25. EMSA- M. hyopneumoniaeHrcA-CIRCE Interaction 1 2 3 4 5

  26. DNaseI Footprinting –JBBM 30 (1995) 85-89

  27. DNA Affinity Chromatography

  28. Surface Plasmon Resonance (SPR) • SPR - Surface plasmon resonance is a phenomenon which occurs when light is reflected off thin metal films. A fraction of the light energy incident at a sharply defined angle can interact with the delocalized electrons in the metal film (plasmon) thus reducing the reflected light intensity

  29. DNA-Binding Motif Comparison of aa sequences of several leucine zipper proteins

  30. Leucine zipper from yeast activator protein (1YSA)

  31. Helix-loop-helix –the human transcription factor Max, bound to DNA target 1HLO

  32. 2. Regulation of Gene Expression in Prokaryotes

  33. Catabolic Repression - restricts expression of the genes required for catabolism of lactose, arabinose and other sugar in the presence of glucose

  34. CRP (cAMP Receptor Protein) homodimer - bound with cAMP

  35. The trp Operon

  36. The trp Repressor

  37. Transcriptional attenuation in the trp operon

  38. SOS response in E. coli - RecA/ssDNA cleaves repressor LexA

  39. Translational feedback in some ribosomal protein operons Translation Repressor

  40. Stringent response in E. coli –amino acid starvation uncharged tRNA binds to A siteRelA action  ppGpp as starvation signal and regulate ~200 genes and rRNA

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