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Download Nucleoside Phosphoramidate Monoesters: Potential
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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 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. tRNA Activation by aminoacyl tRNA synthetases 1. Aminoacyl-AMP formation: HO O (-)O O P R O O(-) O P C O O +H 3 N O O(-) +H 3 N R P O- Adenine O O C O O Adenine O P O O- + PPi OH OH Aminoacyl adenylate (Aminoacyl-AMP) OH OH 2Pi 2. Aminoacyl transfer to the appropriate tRNA: R R O +H 3 N C O O P O O- Adenine O + HO-ACC-tRNA O +H 3 N C ACC-tRNA + AMP O OH OH Overall reaction: amino acid + tRNA + ATP aminoacyl-tRNA + AMP + PPi 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 Class I and II synthetases bind to different faces of the tRNA molecule Class I synthetases acylate the 2’-OH Class II synthetases acylate the 3’-OH NH2 NH2 tRNA N tRNA N N N O O oo- O N N P O N P O O O O H H H H H H OH O O H O H O OH C C CH CH R NH3 R NH3 N 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. 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. The acylation site of threonyl tRNA synthetase contains a Zinc ion that interacts with the OH group of Threonine O H 2N CH C O OH H 2N CH C CH OH CH CH3 CH3 CH3 Thr Val OH Some amino acids have the same functional groups and differ only by size: O H2N CH C CH CH3 H2C Ile O OH H2N CH C CH CH3 CH3 CH3 Val OH tRNA Synthetase Proofreading •“Double sieve” based on size • Flexibility of the acceptor stem essential Isoleucil-tRNA Synthetase: Proofreading based on size Larger Acylation Site Larger Acylation Site Smaller Hydrolytic Site Smaller Hydrolytic Site CH 3 H3C CH 3 CH 3 O O NH 3 + +H 3 N tRNAIle O CH 3 O tRNAIle Difference in Size H 3C CH 3 O O +H 3 N +H 3 N O CH 3 O tRNAIle Ile Correct Acylation Val Misacylation tRNAIle Valyl tRNAVal Synthetase Proofreading: hydrophobic/polar recognition motif Hydrophobic Acylation Site 3 HC Polar Hydrolytic Site Hydrophobic Acylation Site Polar Hydrolytic Site CH 3 H 3C O OH O +H 3 N NH 3 + O tRNAVal tRNAVal O Difference in Hydrophobicity CH3 CH 3 HO CH 3 O O +H 3 N +H 3 N O tRNAVal Val Correct Acylation O tRNAVal Thr Misacylation 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. 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. Secondary Structure of Transfer RNA molecule 60-93 nt long 7 bp acceptor stem O O H2C H2C NH NH N O dihydrouridine (UH2) HN O pseudouridine ( Examples of tRNA Recognition by aminoacyl tRNA Synthetases tRNAAla 5'P G3 3'OH A C C tRNAPhe 5'P tRNASer 3'OH A C C 5'P U70 C11 A G24 D G34 A35 A36 3'OH A C C Threonyl tRNA synthase complex with tRNA Codon-anticodon recognition between tRNA and mRNA 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 GCU GCC GCA tRNAAla (5’-IGC-3’) alanyl tRNA synthetase 3’ Synthetase 5’ CGI 5’-GCU (C,A)-3’ anticodon codon Genetic Code Codon : Anticodon Recognition 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) 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 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 tRNA Anticodon-Codon Recognition Adenosine Inosine NH 2 O O N N N HN N H N Guanosine N N N HN HN N H N Ribose tRNAAla Anticodon Codon 3' 5' C G NH2 O O I C N C-I base pair 5' 3' 3' 5' C G NH2 G C N C1' C1' N 5' 3' 3' 5' N A-I base pair C G G C 5' 3' I U O N N HN HN I A O N N N HN N C1' G C NH N N C1' C1' O N O HN N N U-I base pair C1' tRNA Anticodon-Codon Recognition Anticodon Codon 3' 5' C G G C 5' 3' I U 3' 5' C G Anticodon Codon 3' 5' C G G C G U 5' Anticodon Codon 3' 5' C G G C U A 5' Anticodon Codon 3' 5' C G G C C G 5' 3' 3' 3' G C I C 5' 3' 3' 5' C G G C G C 5' 3' 5' C G G C U G 5' 3' 5' C G G C A U 5' 3' 5' C G G C 3' 3' 3' I A 5' 3' Genetic Code 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.