Modification of Genes and Proteins

1. Modification of Genes and Proteins By Paul Southard, Joshua Pikovsky, and Jake Secor Do not press any buttons during the presentation, it will progress on its own.

2. Transcript Processing • Protein Folding • RNAi • Gene Repair

3. Transcript Processing

4. Introduction to Transcript Processing • Transcription factor recognizes TATA Box and binds to DNA • RNA polymerase bonds to DNA • RNA polymerase separates strands and strings together complementary nucleotides (using U instead of T) • Primary transcript has been created when terminator region is reached

5. Introduction to Transcript Processing • Transcription: • Creates molecule to carry protein instructions from DNA • Creates exact replica complementary to DNA

6. Transcript Processing • Alteration of ends of transcript: • 5’ end capped with modified guanine • Keeps RNA from degrading in the cytoplasm • Cleavage factors and stabilizing factors bind to 3’ end • Poly A polymerase binds and cleaves 3’ end and adds poly A tail made of adenine

7. Transcript Processing • RNA splicing: • Nucleotides removed • Introns = non-coding regions • Exons = coding regions to be expressed • Small nuclear ribonucleoproteins (snRNPs) = proteins that detect adenine at branching site • Spliceosomes remove the intron and bind the two exons

9. Protein Folding

10. Introduction to Protein Folding • The sequence of amino acids defines a protein’s primary structure.

11. Introduction to Protein Folding • Blueprint for each amino acid is characterized by base triplets • Found in the coding region of genes • Ribosomes recognize triplets and create proteins

13. Protein Folding • Covalent bonds between amino acids help stabilize the protein • Shape and stability also maintained by chemical forces

14. Protein Folding • Chaperone proteins: • Prevent nearby proteins from inappropriately associating and interfering with proper folding • Surround protein in protective chamber during folding • Ex) bacteria: GroEL and GroES • Use ATP • Also assist in refolding proteins

15. Protein Folding Chaperone proteins protecting folding proteins

16. Protein Folding • Models of protein folding: • Diffusion Collision Model: • Nucleus is formed • Secondary structures collide and pack together • Nuclear Condensation Model: • Secondary and tertiary structures are made simultaneously

17. RNAi

18. Introduction to RNAi • RNAi = RNA Interference • Also known as: • Cosuppression • Post Transcriptional Gene Silencing • Quelling • RNAi is used to: • Silence specific genes • Fix gene expression problems in mammals

19. Introduction to RNAi • Types of small silencing RNA: • Small interfering RNA (siRNA) • Endogeneous: derived from cell • Exogeneous: delivered by humans • Micro RNAs (miRNA) • PIWI-interacting RNAs (piRNA) • RNAi breaks up mRNA before it is synthesized.

20. Introduction to RNAi

21. Introduction to RNAi

22. Implications of RNAi • Allows singling out of genes to determine function.

23. Implications of RNAi • Could halt progression of: • Cancer • HIV • Arthritis • All other diseases

24. Implications of RNAi

25. Jean Repair

26. Introduction to Jean Repair

27. Gene Repair

28. Introduction to Gene Repair • DNA can be damaged by: • Radiation (gamma, x-ray, and ultraviolet) • Oxygen radicals from cellular respiration • Environmental chemicals (hydrocarbons) • Chemicals used in chemotherapy

29. Introduction to Gene Repair • Four major types of DNA damage: • Deamination: amino acid group lost • Mismatched base • Backbone break • Covalent cross-linkage between bases Deamination in DNA

30. Gene Repair • Repairing damaged bases: • Direct chemical reversal • Excision repair mechanisms: • Base excision repair (BER) • Nucleotide excision repair (NER) • Mismatch repair (MMR)

31. Gene Repair • Chemical Reversal • Ex) glycosylase enzymes remove mismatched T and restore correct C

32. Gene Repair • Excision repair mechanisms: • Base excision repair: • DNA glycosylases identify damaged bases • DNA glycosylases remove damaged bases • Deoxyribose phosphate backbone component removed, creating gap • Gap filled with correct nucleotide • Break in strand ligated

33. Gene Repair • Excision repair mechanisms: • Nucleotide excision repair: • Protein factors identify damage • DNA is unwound • Faulty area is cut out and the bases are removed • DNA is synthesized to match that of the opposite, correct strand • DNA ligase adds synthesized DNA

34. Gene Repair • Excision repair mechanisms: • Mismatch repair • Corrects mismatches of normal bases (A&T, C&G) by: • Identifying mismatched bases • Cutting mismatched bases

36. Any Questions?