Unit #3 Schedule:

1. Unit #3 Schedule: • Previously: • Sanger Sequencing • Central Dogma Overview • Mutation • Transcription, RNA Processing, Translation • Last Class: • Central Dogma Sculpting • Today: • Regulation of Gene Expression + Trivia • StudyNotes 9 Due • Tutorial (Apr 5) • Review (Apr 9) • EXAM 3(Apr 11) • Homework 6 Due

2. Regulation of Gene Expression There are at least 300 different kinds of cells in the human body. Most of them have identical DNA.

3. Regulation of Gene Expression We examine this at a very general level. Prokaryotes vs. Eukaryotes

4. Definition: Operon • A region of DNA that codes for a series of functionally related genes and is transcribed from a single promoter into mRNA.

5. Negative Control and Positive Control • Transcription can be regulated via negative control or positive control. • Negative control occurs when a regulatory protein binds to DNA and shuts down transcription. • Positive control occurs when a regulatory protein binds to DNA and triggers transcription.

8. Negative Control  Negative control occurs when a regulatory protein binds to DNA and shuts down transcription.

9. Fig. 18-2 PROKARYOTIC REGULATION OF GENE EXPRESSION:

10. Campbell Fig. 18-3a trp operon Promoter Promoter Genes of operon DNA trpD trpR trpA trpE trpC trpB Operator Regulatory gene Stop codon Start codon 3 mRNA 5 RNA polymerase mRNA 5 B A D C E Protein Inactive repressor Polypeptide subunits that make up enzymes for tryptophan synthesis (a) Tryptophan absent, repressor inactive, operon on

11. Fig. 18-3b-2 DNA No RNA made mRNA Protein Active repressor Tryptophan (corepressor) (b) Tryptophan present, repressor active, operon off

14. Fig. 18-4b lac operon lacY DNA lacZ lacI lacA RNA polymerase 3 mRNA mRNA 5 5 Permease Transacetylase -Galactosidase Protein Inactive repressor Allolactose (inducer) (b) Lactose present, repressor inactive, operon on

16. Negative Control • Transcription of Trp Operon in the absence of Tryptophan. • Tryptophan activates the repressor. • Transcription of the Lac Operon in the presence of Lactose. • Lactose deactivates the repressor.

17. Positive Control  Positive control occurs when a regulatory protein binds to DNA and triggers transcription.

18. Fig. 18-5 CAP: catabolite activator protein

19. What is cAMP?

21. mc1r Gene Sequence 5’TGCCCACCCAGGGGCCTCAGAAGAGGCTTCTGGGTTCTCTCAACTCCACCTCCACAGCCACCCCTCACCTTGGACTGGCCACAAACCAGACAGGGCCTTGGTGCCTGCAGGTGTCTGTCCCGGATGGCCTCTTCCTCAGCCTGGGGCTGGTGAGTCTGGTGGAGAATGTGCTGGTCGTGATAGCCATCACCAAAAACCGCAACCTGCACTCGCCCATGTATTCCTTCATCTGCTGTCTGGCCCTGTCTGACCTGATGGTGAGTATAAGCTTGGTGCTGGAGACGGCTATCATCCTGCTGCTGGAGGCAGGGGCCCTGGTGACCCGGGCCGCTTTGGTGCAACAGCTGGACAATGTCATTGACGTGCTCATCTGTGGCTCCATGGTGTCCAGTCTTTGCTTCCTTGGTGTCATTGCCATAGACCGCTACATCTCCATCTTCTATGCATTACGTTATCACAGCATTGTGACGCTGCCCCGGGCACGACGGGCCATCGTGGGCATCTGGGTGGCCAGCATCTTCTTCAGCACCCTCTTTATCACCTACTACAACCACACAGCCGTCCTAATCTGCCTTGTCACTTTCTTTCTAGCCATGCTGGCCCTCATGGCAATTCTGTATGTCCACATGCTCACCCGAGCATACCAGCATGCTCAGGGGATTGCCCAGCTCCAGAAGAGGCAGGGCTCCACCCGCCAAGGCTTCTGCCTTAAGGGTGCTGCCACCCTTACTATCATTCTGGGAATTTTCTTCCTGTGCTGGGGCCCCTTCTTCCTGCATCTCACACTCATCGTCCTCTGCCCTCAGCACCCCACCTGCAGCTGCATCTTTAAGAACTTCAACCTCTACCTCGTTCTCATCATCTTCAGCTCCATCGTCGACCCCCTCATCTATGCTTTTCGGAGCCAGGAGCTCCGCATGACACTCAGGGAGGTGCTGCTGTGCTCCTGGTGA 3’

22. mc1r Gene Sequence 5’TGCCCACCCAGGGGCCTCAGAAGAGGCTTCTGGGTTCTCTCAACTCCACCTCCACAGCCACCCCTCACCTTGGACTGGCCACAAACCAGACAGGGCCTTGGTGCCTGCAGGTGTCTGTCCCGGATGGCCTCTTCCTCAGCCTGGGGCTGGTGAGTCTGGTGGAGAATGTGCTGGTCGTGATAGCCATCACCAAAAACTGCAACCTGCACTCGCCCATGTATTCCTTCATCTGCTGTCTGGCCCTGTCTGACCTGATGGTGAGTATAAGCTTGGTGCTGGAGACGGCTATCATCCTGCTGCTGGAGGCAGGGGCCCTGGTGACCCGGGCCGCTTTGGTGCAACAGCTGGACAATGTCATTGACGTGCTCATCTGTGGCTCCATGGTGTCCAGTCTTTGCTTCCTTGGTGTCATTGCCATAGACCGCTACATCTCCATCTTCTATGCATTACGTTATCACAGCATTGTGACGCTGCCCCGGGCACGACGGGCCATCGTGGGCATCTGGGTGGCCAGCATCTTCTTCAGCACCCTCTTTATCACCTACTACAACCACACAGCCGTCCTAATCTGCCTTGTCACTTTCTTTCTAGCCATGCTGGCCCTCATGGCAATTCTGTATGTCCACATGCTCACCCGAGCATACCAGCATGCTCAGGGGATTGCCCAGCTCCAGAAGAGGCAGGGCTCCACCCGCCAAGGCTTCTGCCTTAAGGGTGCTGCCACCCTTACTATCATTCTGGGAATTTTCTTCCTGTGCTGGGGCCCCTTCTTCCTGCATCTCACACTCATCGTCCTCTGCCCTCAGCACCCCACCTGCAGCTGCATCTTTAAGAACTTCAACCTCTACCTCGTTCTCATCATCTTCAGCTCCATCGTCGACCCCCTCATCTATGCTTTTCGGAGCCAGGAGCTCCGCATGACACTCAGGGAGGTGCTGCTGTGCTCCTGGTGA 3’

23. Consequence of Mutation • A single nucleotide mutation from a Cytosine to a Thymine leads to… • An amino acid change from an Arginine to a Cysteine Amino Acid Sequence Dark Fur: MPTQGPQKRLLGSLNSTSTATPHLGLATNQTGPWCLQVSIPDGLFLSLGLVSLVENVLVVIAITKNRNLHSPMYSFICCLALSDLMVSISLVLETAIILLLEAGALVTRAALVQQLDNVIDVLICGSMVSSLCFLGVIAIDRYISIFYALRYHSIVTLPRARRAIXGIWVASIFFSTLFITYYNHTAVLICLVTFFLAMLALMAXLYVHMLTRAYQHAQGIAQLQKRQGSTXQGFCLKGAXTLTIILGIFFLCWGPFFLHLTLIVLCPQHPTCSCIFKNFNLYLVLIIFSSIVDPLIYAFRSQELRMTLREVLLCSW Amino Acid Sequence Light Fur: MPTQGPQKRLLGSLNSTSTATPHLGLATNQTGPWCLQVSVPDGLFLSLGLVSLVENVLVVIAITKNCNLHSPMYSFICCLALSDLMVSISLVLETAIILLLEAGALVTRAALVQQLDNVIDVLICGSMVSSLCFLGVIAIDRYISIFYALRYHSIVTLPRARRAIVGIWVASIFFSTLFITYYNHTAVLICLVTFFLAMLALMAILYVHMLTRAYQHAQGIAQLQKRQGSTRQGFCLKGAATLTIILGIFFLCWGPFFLHLTLIVLCPQHPTCSCIFKNFNLYLVLIIFSSIVDPLIYAFRSQELRMTLREVLLCSW

24. Changing 1 amino acid: • Arginine: • Strongest +charge • Very hydrophilic • Cysteine: • Not hydrophilic • Forms disulfide bonds

25. Beach Mice • Missense substitution mutation of one nucleotide CT • Changes one amino acid: Arginine  Cysteine • Changes the function of the MC1R protein

26. How is eumelanin produced? • When the MC1R protein is stimulated, cAMP is produced • Lots of cAMP within a melanocyte cell will facilitate the expression of at least four genes:c(tyr), Tyrp1, Tyrp2, p

27. How is eumelanin produced? • When cAMP is plentiful, c(tyr), Tyrp1, Tyrp2 and p are all expressed and their enzymes facilitate the biosynthetic pathway that leads to eumelanin production.

28. How is eumelanin produced? • When cAMP is scarce, c(tyr), Tyrp1, Tyrp2 and p are not as readily expressed. • If only small amounts of cAMP are present, c(tyr) may still be expressed and its enzyme may facilitate the biosynthetic pathway leading pheomelanin production.

29. How is eumelanin produced? • If c(tyr) is not adequately expressed it is possible that neither biosynthetic pigment production pathway may occur. This would result in no pigment production.

30. The Melanocortin-1-Receptor Effectively stimulated by hormone Ineffectively stimulated by hormone MC1RR67 MC1RC67 Results in lots of cAMP production Results in little cAMP production c(tyr), p tyrp1, tyrp2 <<activated>> c(tyr) <<activated>> p, tyrp1, tyrp2 <not activated> LOTS of EUMELANIN produced EUMELANIN not produced

31. Fig. 18-5 CAP: catabolite activator protein

32. EUKARYOTIC REGULATION OF GENE EXPRESSION Activators and Enhancers of Transcription Campbell 8e, Fig. 18.8

34. Fig. 17-8 A eukaryotic promoter includes a TATA box 1 Promoter Template 5 3 3 5 TATA box Template DNA strand Start point Several transcription factors must bind to the DNA before RNA polymerase II can do so. 2 Transcription factors 5 3 3 5 Additional transcription factors bind to the DNA along with RNA polymerase II, forming the transcription initiation complex. 3 RNA polymerase II Transcription factors 3 5 5 5 3 RNA transcript Transcription initiation complex

35. Campbell 8e, Fig. 18.9

37. Controlling Gene Expression: Enhancers and Activators • Provide a way to turn on specific genes in specific cells • Different genes have different enhancers • Different cells have different activators Campbell 8e, Fig. 18.10

38. Controlling Gene Expression: Enhancers and Activators • Tissue- and cell-type specific gene expression • Liver cells make albumin, but not crystallin • Lens cells make crystallin, but not albumin Campbell 8e, Fig. 18.10

39. Coming Up: Friday: • Tutorial (3-5pm in C-3) Tuesday: • Interactive Review (White-Boards + Clickers) Thursday: • Midterm Exam 3

40. Review Part 1 Clicker Review

41. With respect to nucleotide bonds: • A-T is stronger than C-G • C-G is stronger than A-T • A-T and C-G have approximately equal strength

42. Which mode of information transfer usually does not occur? • DNA to DNA • DNA to RNA • DNA to protein • RNA to protein • All occur in a working cell

43. In replication of DNA, the helix is opened and untwisted by • DNA polymerase • ligase • helicase • telomerase • topoisomerase

44. _______________ joins DNA fragments to the lagging strand. (A) Telomere (B) DNA Polymerase I (C) Helicase (D) DNA Polymerase III (E) Ligase

45. In a nucleic acid, the phosphate group, nitrogenous base and free hydroxyl group are attached to the _______________ carbons of ribose (respectively). (A) 1', 3', 5' (B) 5', 3', 1' (C) 3', 5', 1' (D) 5', 1', 3' (E) 3', 1', 5'

46. DNA polymerase III is thought to add nucleotides (A) to the 5' end of the RNA primer (B) to the 3' end of the RNA primer (C) in the place of the primer RNA after it is removed (D) on single stranded templates without need for an RNA primer (E) in the 3' to 5' direction

47. Considering the structure of double stranded DNA, what kinds of bonds hold one complementary strand to the other? • peptide • covalent (C) hydrogen (D) phosphodiester (E) ionic

48. The nitrogenous base adenine is found in all members of which group? • proteins, ATP, and DNA (B) proteins, carbohydrates and ATP (C) glucose, ATP and DNA (D) ATP, RNA and DNA (E) proteins, glycerol and hormones

49. Where and how are Okazaki fragments synthesized? • on the leading strand, in a 5’  3’ direction • on the leading strand, in a 3’  5’ direction • on the lagging strand, in a 5’  3’ direction • on the lagging strand, in a 3’  5’ direction

50. Which of the following types of mutation, resulting in an error in the mRNA just after the AUG start of translation, is likely to have the most serious effect on the polypeptide product? (A) insertion of a codon (B) deletion of two codons (C) substitution of the third nucleotide in an ACC codon (D) deletion of a nucleotide (E) insertion of 9 nucleotides