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Last Class

Last Class. 1. Transcription 2. RNA Modification and Splicing 3. RNA transportation 4. Translation . Quality control of translation in bacteria Rescue the incomplete mRNA process and add labels for proteases. Folding of the proteins Is required before functional.

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Last Class

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  1. Last Class • 1. Transcription • 2. RNA Modification and Splicing • 3. RNA transportation • 4. Translation

  2. Quality control of translation in bacteria Rescue the incomplete mRNA process and add labels for proteases

  3. Folding of the proteins Is required before functional

  4. Folding process starts at ribosome

  5. Protein Folding Pathway Molecular Chaperone

  6. An example of molecular chaperone functions Hsp70, early binding to proteins after synthesis

  7. An example of molecular chaperone functions (chaperonin) Hsp60-like protein, late

  8. The Fate of Proteins after translation

  9. E1: ubiquitin activating enzyme; E2/3: ubiquitin ligase

  10. The production of proteins

  11. Summary • RNA translation (Protein synthesis), tRNA, ribosome, start codon, stop codon • Protein folding, molecular chaperones • Proteasomes, ubiquitin, ubiqutin ligase

  12. Control of Gene Expression • 1. DNA-Protein Interaction • 2. Transcription Regulation • 3. Post-transcriptional Regulation

  13. Neuron and lymphocyte Different morphology, same genome

  14. Six Steps at which eucaryotic gene expression are controlled

  15. Regulation at DNA levels Double helix Structure

  16. The outer surface difference of base pairs without opening the double helix Hydrogen bond donor: blue Hydrogen bond acceptor: red Hydrogen bond: pink Methyl group: yellow

  17. DNA recognition code

  18. One typical contact of Protein and DNA interface In general, many of them will form between a protein and a DNA

  19. DNA-Protein Interaction • Different protein motifs binding to DNA: Helix-turn-Helix motif; the homeodomain; leucine zipper; helix-loop-helix; zinc finger • Dimerization approach • Biotechnology to identify protein and DNA sequence interacting each other.

  20. Helix-turn-Helix C-terminal binds to major groove, N-terminal helps to position the complex, discovered in Bacteria

  21. Homeodomain Protein in Drosophila utilizing helix-turn-helix motif

  22. Zinc Finger Motifs Utilizing a zinc in the center An alpha helix and two beta sheet

  23. An Example protein (a mouse DNA regulatory protein) utilizing Zinc Finger Motif

  24. Three Zinc Finger Motifs forming the recognition site

  25. A dimer of the zinc finger domain of the glucocorticoid receptor (belonging to intracellular receptor family) bound to its specific DNA sequence Zinc atoms stabilizing DNA-binding Helix and dimerization interface

  26. Beta sheets can also recognize DNA sequence (bacterial met repressor binding to s-adenosyl methionine)

  27. Leucine Zipper Dimer Same motif mediating both DNA binding and Protein dimerization (yeast Gcn4 protein)

  28. Homodimers and heterodimers can recognize different patterns

  29. Helix-loop-Helix (HLH) Motif and its dimer

  30. Truncation of HLH tail (DNA binding domain) inhibits binding

  31. Six Zinc Finger motifs and their interaction with DNA

  32. Gel-mobility shift assay Can identify the sizes of proteins associated with the desired DNA fragment

  33. DNA affinity Chromatography After obtain the protein, run mass spec, identify aa sequence, check genome, find gene sequence

  34. Assay to determine the gene sequence recognized by a specific protein

  35. Chromatin Immunoprecipitation In vivo genes bound to a known protein

  36. Summary • Helix-turn-Helix, homeodomain, leucine zipper, helix-loop-helix, zinc-finger motif • Homodimer and heterodimer • Techniques to identify gene sequences bound to a known protein (DNA affinity chromatography) or proteins bound to known sequences (gel mobility shift)

  37. Gene Expression RegulationTranscription

  38. Tryptophan Gene Regulation (Negative control) Operon: genes adjacent to each other and are transcribed from a single promoter

  39. Different Mechanisms of Gene Regulation

  40. The binding site of Lambda Repressor determines its function Act as both activator and repressor

  41. Combinatory Regulation of Lac Operon CAP: catabolite activator protein; breakdown of lactose when glucose is low and lactose is present

  42. The difference of Regulatory system in eucaryotes and bacteria • Enhancers from far distance over promoter regions • Transcription factors • Chromatin structure

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