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tRNA Activation (charging) by aminoacyl tRNA synthetases

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. Aminoacyl-tRNA Synthesis. Summary of 2-step reaction:

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tRNA Activation (charging) by aminoacyl tRNA synthetases

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  1. 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

  2. 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.

  3. 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

  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: • 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

  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 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.

  8. 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.

  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 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

  13. Valyl 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

  14. 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.

  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. tRNAMet), stem sequences can (e.g. tRNAAla), both stem regions and anticodon (e.g. tRNAGln), 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 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

  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: Codon tRNA Synthetase GCU GCC tRNAAla (5’-IGC-3’) alanyl tRNA synthetase GCA 3’ 5’ CGIanticodon 5’-GCU (C,A)-3’codon

  21. Genetic Code

  22. Codon : Anticodon Recognition • 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) 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

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

  24. 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 tRNAAla 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'

  25. 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'

  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.

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