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Posttranscriptional Regulations

Last Class: Gene Regulation 1. DNA-protein interaction, different motifs, techniques to study DNA-protein interaction 2. Gene regulation on DNAs, gene activators (acting on promoter, enhancer, chromatin etc), repressors 3. regulation of gene activators/repressors 4. integrated response. .

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Posttranscriptional Regulations

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  1. Last Class: Gene Regulation1. DNA-protein interaction, different motifs, techniques to study DNA-protein interaction2. Gene regulation on DNAs, gene activators (acting on promoter, enhancer, chromatin etc), repressors3. regulation of gene activators/repressors4. integrated response.

  2. Posttranscriptional Regulations

  3. Posttranscriptional regulation possibilities

  4. Alternative RNA Splicing

  5. Alternative Splicing of RNA of the Drosophila DSCAM gene (axon guidance receptors directing growth cone localization)

  6. Negative and Positive Controls of Alternative Splicing (active regulation)sometimes, the splicing sequence is ambiguous, so constitutive random splicing

  7. Antibodies (membrane-bound, secreted) in B lymphocytecleavage regulated RNA processing (CstF)

  8. RNA Editing (inserting Uracil at different sites and change encoding sequence)

  9. Nuclear ExportRev binds to rev response elelent (RRE), which binds to nuclear export receptor (exportin 1) to regulate nuclear export even without splicinga delay of virus infection symptom

  10. Localization in cytoplasmalso determine the fate of RNAs3’ UTR (untranslated region)

  11. 3’ UTR in regulating LocalizationRed: intact 3’ UTR,Green: 3’ UTR deleted

  12. Translational Regulation

  13. Negative Translational Controlaconitase inhibits ferritin production

  14. Phosphorylation RegulationeIF-2B serving as GEF for eIF-2 and promote translation initiationphosphorylation locked eIF-2 in inactive form

  15. Internal Ribosome Entry Site (IRES)

  16. Two Mechanisms of mRNA decay1. regular 3’ tail shortening followed by ‘5 decapping and degradation2. endonucleolytic cleavage and fast decapping and degrading

  17. The competition between mRNA translation and decayinitiation machinery and deadenylation proteins are all associated with 5’ and 3’

  18. With Iron, aconitase release enhances the production of ferritin to bind iron while destabilizes transferrin receptor mRNA to reduce the transportation of more iron intracellularly

  19. Stop Codon CheckingShould after all the exons (nonsense-mediated mRNA decaying)

  20. SiRNA MechanismRNase, ATP hydrolysis and RNA helicase

  21. Summary Premature termination Alternative RNA Splicing 3’ cleavage and Poly A’ addition RNA editing Necleus transportation Localization of RNA at the cytoplasm Translational initiation Degradation

  22. Cell Membranes • Lipid Bilayers • Cell Membrane

  23. Cell Membrane Views

  24. Phospholipid Molecule

  25. Hydrophilic and Hydrophobic Molecules interacting differently with water

  26. Wedge-shaped lipids form micelles Cylinder-shaped lipids form bilayers

  27. Spontaneous sealing of phospholipid bilayer

  28. Liposomes Proved the self sealing process

  29. Phospholipid mobility Lateral diffusion: Diffusion coefficient 10-8 cm2/sec Migrate in seconds to cover the whole surface Flip-Flop: phospholipid translocators

  30. Cis-double bonds affect packing Saturated: packed and thick Unsaturated : loose and thin Phase transition: liquid to solid

  31. Cholesterol and Glycolipids

  32. Function of Cholesterol • Provide structural support, prevent small molecule to pass • Prevent tight packing and transition

  33. Phospholipid types

  34. Microdomains on plasma membrane Lipid rafts (~ 50nm) Choresterol Sphingolipids (long saturated chains) Other proteins

  35. Asymmetrical distribution of phospholipids and glycolipids Protein kinase C (PKC) binds to negatively charged phosphotidylserine to be functional

  36. Phospholipids in cell signaling PKC Calcium GEF, AKT, migrating front

  37. Phospholipase Cleavage sites

  38. Phosphotidylserine exposed on outer surface as apoptosis signal • Phospholipid translocator • Scramblase

  39. Glycolipids Gm1 Ganglioside with charge can serve as signal for lipid rafts, binds to Cholera toxin

  40. Summary Lipid molecules: phospholipids, cholesterol, glycolipid, all amphipathic Lipid bilayer, hydrophobic inside and hydrophilic outside Subdomains on membrane, asymmetry important for functions Phospholipids as signals

  41. Membrane Proteins Integral membrane protein Peripheral membrane protein Glycosylphosphatidylinositol (GPI) anchor protein

  42. Fatty acid chain (acyl, N-terminal) or prenyl group modifications (C-terminal) Or geranylgeranyl Or palmitic acid

  43. Hydropathy Plots Index of hydrophobicity

  44. Membrane proteins are glycosylated Sugar are added in the lumen of the ER and Golgi apparatus, therefore, sugar are outside of cell surface Cytosol has reduced environment, preventing disulfide bonds

  45. Detergent to solubilize and purify membrane proteins A detergent micelle

  46. Solubilize membrane proteins with detergent

  47. Different Detergents Ionic (strong) or nonionic (weak)

  48. Mild detergent for the solubilizing, purification, and reconstitution of membrane protein functions to study the functions of membrane proteins in simplified environment

  49. The study of membrane proteins Most prominent example red blood cells No nucleus or internal organelles

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