Hebrews 1:1-2

1. Hebrews 1:1-2 1 God, who at sundry times and in divers manners spake in time past unto the fathers by the prophets, 2 Hath in these last days spoken unto us by his Son, whom he hath appointed heir of all things, by whom also he made the worlds;

2. TranscriptionFrom DNA To RNA Timothy G. Standish, Ph. D.

3. DNA Transcription Ribosome mRNA Translation Polypeptide (protein) IntroductionThe Central Dogma of Molecular Biology Cell

4. RNA Polymerase • RNA Polymerase is a spectacular enzyme, functioning in: • Recognition of the promoter region • Melting of DNA (Helicase + Topisomerase) • RNA Priming (Primase) • RNA Polymerization • Recognition of terminator sequence

5. Transcription Start Site 3’ Untranslated Region 5’ Untranslated Region 5’ Protein Coding Region 3’ RNA Transcript Promoter/ Control Region Terminator Sequence A “Simple” Gene

6. Stages of Transcription • Initiation • Elongation • Termination

7. Prokaryotic Transcription Initiation • The a subunit of prokaryotic RNA polymerase is necessary for promoter recognition and binding of RNA polymerase to the promotor • Different a subunits allow recognition of different types of promoters thus the type of genes transcribed can be modulated by altering the types of a subunits which attach to RNA polymerase

8. P2 Different promoters Prokaryotic Transcription Initiation RNA Pol.  P1 Heat Shock Gene Constitutive Gene

9. Prokaryotic Transcription Initiation RNA Pol.  P1 Heat Shock Gene P2 Constitutive Gene Different promoters

10. Eukaryotic Transcription Initiation • Proteins called transcription factors bind to the promoter region of a gene • If the appropriate transcription factors are present, RNA polymerase binds to form an initiation complex • RNA polymerase melts the DNA at the transcription start site • Polymerization of RNA begins

11. Promoter T. F. RNA Pol. T. F. RNA Pol. RNA 5’ Initiation T. F.

12. Promoter T. F. RNA Pol. RNA Pol. RNA 5’ Initiation T. F. T. F.

13. Transcription Termination There are two types of termination: • Rho dependent requires a protein called Rho, that binds to and slides along the RNA transcript. The terminator sequence slows down the elongation complex, Rho catches up and knocks it off the DNA • Rho independent termination depends on both slowing down the elongation complex, and an AT-rich region that destabilizes the elongation complex

14. RNA Pol. RNA Pol. RNA 5’ RNA 5’ TerminationRho Independent Terminator

15. RNA Pol. RNA Pol. 5’ RNA Terminator RNA 5’ TerminationRho Independent

16. RNA Pol. r r RNA Pol. RNA 5’ Terminator RNA 5’ TerminationRho Dependent The terminator sequence slows RNA polymerase

17. RNA Pol. r Help, Rho hit me! RNA Pol. RNA 5’ r Terminator RNA 5’ TerminationRho Dependent Rho catches up with RNA polymerase

18. RNA Pol. r r RNA Pol. 5’ RNA Terminator RNA 5’ TerminationRho Dependent The elongation complex disintegrates

19. DifferencesBetween Transcription InProkaryotes and Eukaryotes

20. 5’ 3’ 3’ 5’ RNA Pol. Ribosome mRNA Ribosome 5’ Transcription And Translation In Prokaryotes

21. Cytoplasm Nuclear pores AAAAAA AAAAAA DNA Transcription RNA RNA Processing G G mRNA Export Nucleus Eukaryotic Transcription

22. Transcription Start Site 3’ Untranslated Region 5’ Untranslated Region Introns 5’ 3’ Int. 1 Int. 2 Exon 1 Exon 2 Exon 3 Promoter/ Control Region Terminator Sequence Exons RNA Transcript 5’ 3’ Int. 1 Int. 2 Exon 1 Exon 2 Exon 3 A “Simple” Eukaryotic Gene

23. 5’ Untranslated Region 3’ Untranslated Region 5’ 3’ G AAAAA Exon 1 Exon 2 Exon 3 5’ Cap 3’ Poly A Tail 5’ 3’ Int. 1 Int. 2 Exon 1 Exon 2 Exon 3 Int. 1 Int. 2 Processing Eukaryotic mRNA Protein Coding Region • RNA processing achieves three things: • Removal of introns • Addition of a 5’ cap • Addition of a 3’ tail • This signals the mRNA is ready to move out of the nucleus and may control its lifespan in the cytoplasm

24. Intron Exon 1 Exon 2 GU A AG 5’ 3’ 18-40 BP Left (donor) 5’ splice site Right (acceptor) 3’ splice site Branch site U A C U A A C (Yeast) Common Splicing Mechanism Py80NPy80Py87Pu75APy95 (Animal-Subscripts indicate percent frequency) The branch sequence allows identification of the 3’ splice site

25. Common Splicing Mechanism Yee ha! Intron Exon 2 A AG 5’ 3’ 3’ G U Lariat Lariat l l Exon 1 Lariat Formation

26. A AG G U Intron lariat Common Splicing Mechanism Exon 1 Exon 2 5’ 3’ Following excision, the lariat is rapidly degraded

27. Common Splicing Mechanism Exon 1 Exon 2 5’ 3’ Following excision, the lariat is rapidly degraded

28. The Spliceosome • Spliceosomes are structures that form within the nucleus to remove introns from eukaryotic hnRNA • This structure is large, on the order of a ribosome subunit • Like the ribosome, spliceosomes are composed of both protein and RNA

29. Wobble Base Pairing

30. The Rules of Codon Anticodon Base Pairing • Three things affect the way in which base pairing occurs between codons on mRNA and anticodons on tRNA: • How the two molecules “twist” when annealing - They are not free to form a perfect A helix • The environment of the Ribosome A site • Chemical modification of bases • These three factors change the usual base pairing seen in DNA and RNA, particularly at the first base of anticodons/third base of codons

31. 3’ A Acceptor Arm - A specific amino acid is attached to the 3’ end C C TyC arm - y stands for pseudouridine 73 5’ G C 2 71 3 70 4 69 5 68 6 67 59 7 66 Py A* U* 65 64 63 62 C 16 Pu 17 9 A Pu 17:1 13 12 Py 10 49 50 51 52 G C T y G* Py 22 23 Pu 25 47:16 G A 26 47:15 20 20:2 20:1 27 1 43 44 28 42 45 D Arm - Contains dihydrouridine 46 29 41 Extra Arm - May vary in size 47 30 40 47:1 31 39 Py* 38 U Pu* 34 36 35 Anticodon Transfer RNA (tRNA)

32. O Dihydro-uridine A C C TyC arm - y stands for pseudouridine H 73 NH G C H 2 71 H 3 70 H 4 69 N O 5 68 6 67 59 7 66 Py A* U* 65 64 63 62 C 16 Pu 17 9 A Pu 17:1 13 12 Py 10 49 50 51 52 G C T y G* Py O 22 23 Pu 25 47:16 G Pseudo-uridine A 26 47:15 20 20:2 20:1 27 1 43 44 28 42 45 D Arm - Contains dihydrouridine 46 29 41 47 30 40 HN NH 47:1 31 39 Py* 38 U Pu* 34 N N O 36 35 Anticodon Transfer RNA (tRNA)

33. Some Other Strange tRNA Bases

34. Cytosine N O O N N N N Guanine H H O N H N N N H H Base PairingGuanine And Cytosine - + + - - +

35. H H + N Adenine - - Uracil N N N O O N N + N N H Base PairingAdenine And Uracil

36. + H H + N - Adenine - - Cytosine N O N N N O N H H N N N Base PairingAdenine And Cytosine

37. - + + - Uracil N O O N N N N Guanine O + N N H H N H H Base PairingGuanine And Uracil

38. - + + Uracil + N O O N N N N Guanine O - N N H H N H H Wobble Base PairingGuanine And Uracil

39. H H + N Adenine 2 Thio- uracil - - N N N O N N S + N N H Base PairingAdenine And 2-Thiouracil

40. - + O + H N N N N N Guanine N O S H N H H Wobble Base PairingGuanine And 2-Thiouracil 2 Thio- uracil + 2-Thiouracil forms only one hydrogen bond with guanine which is not enough to form a stable pair in the environment of the ribosome A site

41. - O + Cytosine N N N N N O O Inosine H H + - N H H - N N Wobble Base PairingInosine And Cytosine

42. - Uracil O + O N N O N N N Inosine + - H N N H H Wobble Base PairingInosine And Uracil

43. H - O + Adenine N H N N N N N Inosine + - N N H N H Wobble Base PairingInosine And Adenine

44. The Wacky Rules ofWobble Base Pairing First anticodon base: Third codon base: ---------- A or G ----- A ---------- G ---------- U ---------- C or U ---------- C U or G • U • 2-S-U • C • A • G • I

45. Wobbling and tRNA Numbers • The net effect of wobble base pairing is to reduce the number of tRNAs that must be produced by a cell • In reality cells do not make 61 different tRNAs, one for each codon • Many tRNAs have anticodons that anneal to several different codons\ • Codons are known for which there are more than one tRNA, although each tRNA carries the same amino acid (i.e., methionine)

46. The End