Formation of RNA Polymerase II pre-initiation complex

1. Formation of RNA Polymerase II pre-initiation complex IID contains TBP that binds TATA box IIA stabilizes IID binding to promoter IIB binds initiation sequence Pol II binds IIB IIE stimulates transcription IIH has kinase and helicase activity

2. RNA Synthesis in E. Coli Transcription bubble

4. RNA splicing in eukaryotes Primary transcript, hnRNA

5. Alternative splicing patterns give rise to multiple proteins from the same pre-mRNA

6. RNA Synthesis: Take Home Message 1) DNA sequences are translated into RNA messages by RNA polymerases. 2) The initiation of RNA synthesis is controlled by specific DNA promoter sequences. 3) The synthesis of RNA is governed by initiation, elongation, and termination steps. 4) Eukaryotic mRNA is extensively processed

7. Overview of Protein Synthesis (Translation). Transfer RNA. Required reading: Stryer’ Biochemistry 5th edition Ch. 5, p. 132-136, Ch. 28 p. 797, Ch. 29 p. 813-823 or Stryer 4th edition p. 102-104, 109-112, Ch. 34, p. 875-888 and Ch. 33 p. 849-850

8. Flow of Genetic Information DNA RNA Proteins Cellular Action replication transcription translation DNA

10. How do we go from mRNA to Protein? mRNA Protein DNA t-RNA Amino Acid Sequence

11. Transfer RNA • Acts as an adaptor molecule between mRNA and peptide sequence • Contains amino acid attachment site and template recognition site aminoacyl tRNA synthetase

12. Translation of mRNA 3’ 5’ N C • mRNA is read sequentially from a fixed starting point • Sequence of 3 Bases = One Codon (no gaps) • One Codon = One Amino Acid • 20 Natural Amino Acids = 64 Codons (43) • “Degenerate Code: Several Different Codons per Amino Acid”

13. Genetic Code

14. During translation, mRNA passes through the ribosome so that each codon recognizes its tRNA

15. Translating the Message • DNA 5'-ATG-GCC-TTT-GAT-TCT-AAA-TAA-3' RNA 5'-AUG-GCC-UUU-GAU-UCU-AAA-UAA-3' Protein N- met ala phe asp ser lys stop -C Correct reading frame is essential

16. Write the sequence of amino acids in a polypeptide translated from the following mRNA: 5’ GGA GGA GUA AGU UGU Gly – Gly – Val – Ser - Cys

17. The genetic code is nearly universal, with the exception of mitochondria

18. Transfer RNA

19. Secondary Structure of Transfer RNA molecule 60-93 nt long 7 bp acceptor stem

20. Base sequence of yeast tRNAAla: cloverleaf folding result from the presence of “self complementary” regions -UCCGGTCGAUUCCGGA-

21. tRNA has an “L” Tertiary Structure T Loop Acceptor Stem Amino acid attachment site D Loop V Loop Anti-Codon

22. Tertiary base pairs are responsible for the tertiary structure of tRNA Non-standard H bond interactions, some linking 3 bases, help stabilize the L-shaped tertiary structure of tRNA.

23. tRNA Formation in E. Coli • 60 genes for tRNA are clustered in 25 transcription units • tRNA Precursors containing several tRNAs are cleaved with RNases: RNase P RNase P 3' CCA 5' CCA RNase D RNase D RNase P – generates 5’ ends of tRNA by cleaving the bond 5’ to each tRNA RNase D – trims the 3’ end up to the CCA sequence

24. tRNA Activation (charging) by aminoacyl tRNA synthetases Aminoacyl tRNA synthetase • Two important functions: • Implement genetic code • Activate amino acids for • peptide bond formation • The key enzymes: • Amanoacyl-tRNA synthetases

25. Aminoacyl-tRNA Synthesis Summary of 2-step reaction: 1. amino acid+ ATPaminoacyl-AMP + PPi 2. aminoacyl-AMP+tRNAaminoacyl-tRNA+ AMP The 2-step reaction is spontaneous overall, because concentration of PPi is kept low by its hydrolysis, catalyzed by Pyrophosphatase.

26. tRNA Activation by aminoacyl tRNA synthetases +H N 3 +H N 3 Aminoacyl adenylate (Aminoacyl-AMP) +H N +H N 3 3 1. Aminoacyl-AMP formation: HO O R (-)O P O O O(-) O O P Adenine + PPi C P R O O O O- O Adenine O O(-) P O C O O- O OH OH 2Pi OH OH 2. Aminoacyl transfer to the appropriate tRNA: R R O O O Adenine HO-ACC-tRNA + ACC-tRNA + AMP C P C O O O- O O OH OH Overall reaction: amino acid + tRNA + ATP  aminoacyl-tRNA + AMP + PPi

27. Classes of Aminoacyl-tRNA Synthetases • • Class I: Arg, Cys, Gln, Glu, Ile, Leu, Met, Trp, Tyr, Val • (Generally the Larger Amino Acids) • • Class II: Ala, Asn, Asp, Gly, His , Lys, Phe, Ser, Pro, Thr • (Generally the smaller amino acids) • Main Differences between the two classes: • Structural differences. Class I are mostly monomeric, • class II are dimeric. • Bind to different faces of the tRNA molecule • 3. While class I acylate the 2’ hydroxyl of the terminal Ado, • class II synthetases acylate the 3’-OH

28. Class I and II synthetases bind to different faces of the tRNA molecule

29. Class I synthetases • acylate the 2’-OH • Class II synthetases • acylate the 3’-OH

30. The accuracy of protein synthesis depends on correct • charging of tRNAswith amino acids • tRNA synthetases must link tRNAs with their correct amino • acids. • 2. tRNA synthetases recognize correct amino acids by specific • binding to the active site and proofreading. • 3. tRNA synthetases recognize correct tRNAs via by interacting with • specific regions of tRNA sequence.

31. The accuracy of protein synthesis depends on correct • charging of tRNAswith amino acids • tRNA synthetases must link tRNAs with their correct amino • acids. • 2. tRNA synthetases recognize correct amino acids by specific • binding to the active site and proofreading. • 3. tRNA synthetases recognize correct tRNAs via by specific • regions of tRNA sequence.

32. The acylation site of threonyl tRNA synthetase contains a Zinc ion that interacts with the OH group of Threonine

33. Threonyl-tRNA synthetase contains editing site

34. tRNA Synthetase Proofreading • “Double sieve” based on size • Flexibility of the acceptor stem essential

35. tRNA Synthetase Proofreading Larger Larger Smaller Smaller Acylation Site Acylation Site Hydrolytic Site Hydrolytic Site CH 3 H C CH CH 3 3 3 NH + +H N 3 3 CH 3 H C CH 3 3 CH 3 +H N 3 +H N 3 O O O O tRNAIle tRNAIle O O O tRNAIle O tRNAIle Val Ile Misacylation Correct Acylation

36. tRNAVal Synthetase Proofreading: hydrophobic/polar recognition motif Hydrophobic Polar Hydrophobic Polar Acylation Site Hydrolytic Site Acylation Site Hydrolytic Site HC CH H C 3 3 3 +H N NH + 3 3 CH3 CH 3 CH 3 +H N 3 +H N 3 OH O O O tRNAVal O tRNAVal Difference in Hydrophobicity HO O O O tRNAVal O tRNAVal Val Thr Correct Acylation Misacylation

37. The accuracy of protein synthesis depends on correct • charging of tRNAswith amino acids • tRNA synthetases must link tRNAs with their correct amino • acids. • 2. tRNA synthetases recognize correct amino acids by specific • binding to the active site and proofreading. • 3. tRNA synthetases recognize correct tRNAs via using specific • regions of the tRNA sequence.

38. tRNA Recognition by Synthetases • different recognition motif depending on synthetase • usually just a few bases are involved in recognition • Can involve specific recognition of the anticodon (e.g. tRNAMet), stem sequences can (e.g. tRNAAla), both stem regions and anticodon (e.g. tRNAGln), or, less frequently, D loop or T loop bases.

39. A A A C C C C C C Examples of tRNA Recognition by aminoacyl tRNA Synthetases tRNAAla tRNAPhe tRNASer 3' OH 3' OH 3' OH 5' P 5' P 5' P G3 U70 C11 A G24 D G34 A36 A35

40. Threonyl tRNA synthase complex with tRNA

41. Codon-anticodon recognition between tRNA and mRNA

42. The relationship between the number of codons, tRNAs, and synthetases Total of 61 codons, but not 61 tRNAs! The same tRNA can recognize more than one codon Example: Codon tRNA Synthetase GCU GCC tRNAAla (5’-IGC-3’) alanyl tRNA synthetase GCA 3’ 5’ CGIanticodon 5’-GCU (C,A)-3’codon

43. Codon : Anticodon Recognition • The first two interactions (XY-X’Y’) obey Watson-Crick • base pairing rules. • 2. The third interaction is less strict (Wobble pairing is allowed) 3 2 1 t RNA- 3'-X Y Z -5' anticodon mRNA- 5'-X’Y’Z’-3' codon 1 2 3 The Third Base of Codon is Variable

44. Wobble base pairing rules 5’ anticodon base 3’ codon base C G A U U A or G G C or U I U, C, or A

45. tRNA Anticodon-Codon Recognition NH 2 N N H H Adenosine Guanosine Inosine O O N N N HN N HN N N HN N N Ribose 5' 5' 5' Anticodon 3' C G I 3' C G I 3' C G I Codon 5' G C C 3' 5' G C A 3' 5' G C U 3'

46. tRNA Anticodon-Codon Recognition 5' 5' 5' Anticodon 3' C G I 3' C G I 3' C G I Codon 5' G C U 3' 5' G C C 3' 5' G C A 3' 5' 5' Anticodon 3' C G G 3' C G G Codon 5' G C U 3' 5' G C C 3' 5' 5' Anticodon 3' C G U 3' C G U Codon 5' G C A 3' 5' G C G 3' 5' 5' Anticodon 3' C G C 3' C G A Codon 5' G C G 3' 5' G C U 3'

47. Genetic Code

48. Nonsense suppression Nonsense mutations = change of codon for an aa to STOP Usually lethal – truncated protein Can be rescued by mutation in a different part of the genome Mechanism: tRNA gene mutation Example: E. Coli Amber suppressor tRNATyr anticodon change GUA  CUA Mutated tRNA recognized stop codon as Tyr and prevents chain termination

49. Overview of Protein Synthesis : Take Home Message 1) Translation of the genetic code is dependent on three base words that correspond to a single amino acid. 2) The mRNA message is read by tRNA through the use of a three base complement to the three base word. 3) A specific amino acid is conjugated to a specific tRNA (three base word). 4) Amino acid side chain size, hydrophobicity and polarity govern the ability of tRNA synthetases to conjugate a specific three base message with a specific amino acid.