1. tRNA Activation (charging) by aminoacyl tRNA synthetases Aminoacyl tRNA synthetase Two important functions: 1.Implement genetic code 2.Activate amino acids for peptide bond formation The key enzymes: Amanoacyl-tRNA synthetases

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

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

4. 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: 1.Structural differences. Class I are mostly monomeric, class II are dimeric. 2.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

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

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

7. The accuracy of protein synthesis depends on correct charging of tRNAs with amino acids 1.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.

8. The accuracy of protein synthesis depends on correct charging of tRNAs with amino acids 1.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.

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

10. Some amino acids have the same functional groups and differ only by size:

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

12. Isoleucil-tRNA Synthetase: Proofreading based on size CH 3 O +H 3 N tRNA Ile O CH 3 Smaller Hydrolytic Site Larger Acylation Site CH 3 H 3 C O NH 3 + O Larger Acylation Site Smaller Hydrolytic Site tRNA Ile CH 3 O +H 3 N tRNA Ile O CH 3 Correct Acylation H 3 CCH 3 O +H 3 N tRNA Ile O Misacylation Ile Val

13. Valyl tRNA Val Synthetase Proofreading: hydrophobic/polar recognition motif 3 HCCH 3 O +H 3 N tRNA Val O Hydrophobic Acylation Site Polar Hydrolytic Site CH 3 CH 3 O +H 3 N tRNA Val O Correct Acylation HOCH 3 O +H 3 N tRNA Val O Misacylation OHH 3 C O NH 3 + O tRNA Val Polar Hydrolytic Site Hydrophobic Acylation Site Difference in Hydrophobicity Val Thr

14. The accuracy of protein synthesis depends on correct charging of tRNAs with amino acids 1.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.

15. 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. tRNA Met ), stem sequences can (e.g. tRNA Ala ), both stem regions and anticodon (e.g. tRNA Gln ), or, less frequently, D loop or T loop bases.

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

17. A OH P5' 3' U70G3 A C C OH P5' 3' A C C OH P5' 3' A C C tRNA Ala tRNA Phe G34 A35 A36 Examples of tRNA Recognition by aminoacyl tRNA Synthetases tRNA Ser C11 G24 D

18. Threonyl tRNA synthase complex with tRNA

19. Codon-anticodon recognition between tRNA and mRNA

20. 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: CodontRNASynthetase GCU GCCtRNA Ala (5’-IGC-3’)alanyl tRNA synthetase GCA CGI anticodon 5’-GCU (C,A)-3’ codon 5’3’

21. Genetic Code

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

23. Wobble base pairing rules first anticodon base (Z) third codon base (Z’) CG AU UA or G GC or U IU, C, or A t RNA- 3'-X Y Z -5' anticodon mRNA- 5'-X’Y’Z’-3' codon 1 2 3 3 2 1

24. tRNA Anticodon-Codon Recognition HN N N N O Inosine Ribose N N N H N NH 2 HN N N H N O Adenosine Guanosine CGI GCC CGI GCA CGI GCU Codon Anticodon 5' 3' 5' 3' 5' 3' 5' tRNA Ala

25. tRNA Anticodon-Codon Recognition CGI GCU CGI GCC CGI GCA Codon Anticodon 5' 3' 5' 3' 5' 3' 5' CGG GCU CGG GCC Codon Anticodon 5' 3' 5' 3' 5' CGU GCA CGU GCG Codon Anticodon 5' 3' 5' 3' 5' CGC GCG CGA GCU Codon Anticodon 5' 3' 5' 3' 5'

26. Genetic Code

27. 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.