Modification of Genes and Proteins

1. Modification of Genes and Proteins By Paul Southard, Joshua Pikovsky, and Jake Secor

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?