2. This Week Chapters 9, 10.1 and 10.4 - 10.7 for reference, –exam material will be on lecture content for the above, –sample questions, and questions for these chapters will be posted this afternoon. Exam Friday: Assignments in Chapters 6, 11, 8, 9 and 10. All lecture material.

3. Gene Expression …the processes by which information contained in genes and genomes is decoded by cells,...in order to produce molecules that determine the phenotypes observed in organisms, –transcription (post-transcriptional modifications), –translation (post-translational modifications.

4. Transcription...the synthesis of mRNA from a DNA template, - now it is important to understand when and where, as well as how.

5. mRNA Synthesis Template (DNA) and Promoter, Nucleoside triphosphates (NTPs), –N: A,U,G,or C, Enzymes (RNA polymerases), Energy (as in replication, from phosphate bonds).

6. E. coli RNA Polymerase

7. RNA Polymerase Scanning

8. E. coli Promoter Sequences

9. Promoter Regions Core Promoter ( ~ 0 to ~ -40 bp)Proximal ( ~ 100 - 200)

10. Regulation of Transcription (Prokaryotes) Regulation of gene expression is often at the transcription level, –Negative regulation, inducible, repressible, –Positive regulation Gene native state is “on”. Gene native state is “off”.

11. Negative Regulation (Inducible) …default state is “on”, i.e., the gene is transcribed. Protein! Protein, or metabolite, etc.

12. Negative Regulation (Repressible) …default state is “on”, i.e., the gene is transcribed. Protein! Protein, or metabolite, etc.

13. Positive Regulation …default state is “off”, i.e., the gene is not transcribed.

14. Prokaryote Review

15. TATA-binding protein. Basal Factors TFIIA binds to the TFIID complex to block inhibitors (D-A complex). TFIIB binds to the D-A complex. RNA polymerase II and TFIIF complex bind. TFIIE, TFIIH and THFIIJ added in order. > 8 sub-units

16. Eukaryotic Initiation

17. Promoter Bashing Eukaryotic Enhancers and Silencers can act at great distances.

18. Drosophila Guts and Such dpp locus...(c) and (d) are ID enhancer driven, give rise to fly appenages.

19. Terms cis-acting elements; –DNA sequences that serve as attachments sites for the DNA- binding proteins that regulate the initiation of transcription. trans-acting elements; –the DNA-binding proteins that regulate the initiation of transcription.

20. Chromatin Remodeling Expressed Proteins

21. Alternate Promoters (Figure 10.22)

22. Transcription Unit …the portion of a gene that specifically codes for a protein (cistron), plus other mRNA. 5’5’ 3’3’ transcription unit RNA polymerase begins transcription here, just “upstream” of the DNA that codes for the protein.

23. Untranslated Regions UTRs are transcribed, but not translated. 5’ UTR 3’ UTR UTR sequences diverge more rapidly during evolution than structural regions.

24. Terminator …a sequence of nucleotides (AAUAAA in the transcribed molecule) that specifies the end of the transcription unit. 5’5’ 3’3’ transcription unit RNA polymerase begins transcription here. Terminator

25. RNA Synthesis …from one strand of the double helix, DNA template strand is read 3 ’ to 5 ’, RNA strand ‘grows from 5 ’ to 3 ’,

26. Elongation Nucleotides are added to the 3 ’ end of the elongating RNA.

28. mRNA vs. pre-mRNA prokaryotic mRNA synthesis described so far requires little, or no further modification prior to translation into proteins, eukaryotic mRNA requires extensive modifications.

29. Post Transcriptional Modification I Occurs in the nucleus. Increases stability, may help transport and sorting. …modified guanine cap added to the 5’ end. …lots of adenines added to the 3’ end. 5’ UTR 3’ UTR

30. Post Transcriptional Modification II Introns affect expression. Differential splicing can alter the protein’s function. Provides “functional cassettes”, for evolutionary mixing and matching. …intervening sequences (introns), do not code for proteins. …code for proteins.

31. small nuclear Riboproteins (snRNPs) Introns are spliced out at structure called spliceosomes, mRNA remains relatively stable, introns are digested rapidly.

32. Splicosomes snRNP = small nuclear RiboNucleoProtein.

33. Study Figure 9.15 for Wednesday

34. Intron Excision

35. Alternate Excision (example)

36. Genetic Code …RNA is an intermediary in the transfer of information from DNA to the synthesis of protein, …how is that information organized?

37. Code is Linear 5 ’ -AAA GGC TGA TCA ATC GAT CGT GAC-3 ’ The 5 ’ -3 ’ sequence from the DNA template is equivalent to the mRNA (except for uracils). 3 ’ -TTT CCG ACT AGT TAG CTA GCA CTG-5 ’ 5 ’ -AAA GGC UGA UCA AUC GAU CGU GAC-3 ’ DNA code strand... DNA template strand... RNA strand...

38. Information Capacity need code for 20 Amino Acids, two base-pair code, –16 different combinations of 4 bases (4 2 ), aa, at, ac, ag, tt, tc, tg, cc, cg, gg, ta, ca, ga, ct, gt, gc, three base-pair code, –64 combinations of 4 bases (4 3 ).

39. Codons …a triplet of nucleotide bases that specifies or encodes the information for a specific amino acid, –also need codons to indicate the beginning and end of the protein to be synthesized.

40. Revertants returned the code to an “in frame” conditions. rII Again

41. Code is Degenerate 20 amino acid codons + start and stop codons 20-some required 64 possible All combinations are used.

42. Start/Stop Codons …AUG codes for the ‘start of translation’, a methionine, –most proteins thus begin with the amino acid methionine, …UAA, UAG and UGA are stop codons, indicating the C terminus of the protein.

43. Wobble: the third nucleotide is not necessarily specific.

46. Central Dogma DNA RNA Protein transcription replication translation

47. Translation …the synthesis of a polypeptide. This occurs on ribosomes using the information encoded on mRNA, –tRNA molecules mediate the transfer of information between mRNA and the growing polypeptide.

48. tRNA anti-codon: site of interaction with the mRNA template. amino acid attachment site: each amino acid has specific tRNA(s),

49. The ‘Ends’ to the Means Specific anti-codons for specific amino acid designation. “anti” = complementary tRNA secondary structure ‘reveals’ a three base pair structure on one end,

50. activating enzymes: aminoacyl-tRNA synthetases, one for each amino acid.

51. Ribosomes …a supramolecular complex of rRNA and proteins, approximately 18 - 22 nm in diameter, …the site of protein synthesis,

52. Ribosome Structure

53. Structure/Function P site (peptidyl): binds the tRNA that holds the growing polypeptide. A site (amino acyl): binds the tRNA that holds the next amino acid, E site (exit): uncharged tRNAs are discharged from here.

54. Initiation large sub-unit binds the complex, P site associates with the Met- tRNA. small sub-unit and a charged Met- tRNA (methionine) forms the initiation complex,

55. Elongation (3 steps) 1. recognition: tRNA anti-codon matches RNA codon, 2. amino acid(s) from P site tRNA transfered to the new tRNA, peptide bond formed, 3. translocation, tRNAs shift, mRNA shifts 1 codon, leaves open A site, cycle repeats. (uncharged tRNA exits)

56. Peptide Linkage hydrolysis reaction

57. Elongation (3 steps) Peptide bond formed.

58. Termination stop codon attracts a protein release factor, hydrolysis of last tRNA/amino acid yields terminal carboxyl group, ribosome disassembles.

59. N-Terminus --> C-Terminus...polypeptides are synthesized beginning from the N-terminus (amino terminus) and going to the C-terminus (carboxy terminus), …this corresponds to the 5 ’ -3 ’ DNA Coding sequence.

60. Wednesday