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Translation

Translation. Chapter 9. Overview. Occurs on ribosomes-large aggregates of rRNA and protein tRNA acts as amino acid carriers Prokaryotes—occurs simultaneously with transcription and mRNA degradation Eukaryotes—occurs in cytoplasm mRNA translated 5’ 3’ Protein synthesis aminocarboxy.

lillian-schroeder
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Translation

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  1. Translation Chapter 9

  2. Overview • Occurs on ribosomes-large aggregates of rRNA and protein • tRNA acts as amino acid carriers • Prokaryotes—occurs simultaneously with transcription and mRNA degradation • Eukaryotes—occurs in cytoplasm • mRNA translated 5’3’ • Protein synthesis aminocarboxy

  3. Protein Synthesis • Polymerization of amino acids: condensation reaction (dehydration synthesis)

  4. ~Universal Genetic Code • Codons—sets of 3 nucleotides corresponding to a single amino acid • Each codon specifies a single amino acid • More than one codon can specify the same amino acid • code is said to be degenerate • Some aa correspond to a single codon • AUG—initiator codon, methionine (Met, M) • UGG–Tryoptophan (TrP, W) • Often codons encoding the same aa differ onl;y at the 3rd nucleotide

  5. ~Universal Genetic Code

  6. NH3+ H-C-CH2SeH COO- Why~Universal? Exceptions • GUG sometimes used as a start • Mammalian mitochondria • Ciliated protozoa • Selenocysteine

  7. Selenocysteine • The 21st amino acid? • An essential amino acid for selenoproteins EX. Glutathione oxidase • Uses unique tRNA (tRNASec), initially bound to Ser. Longest known tRNA (95nt). • Anticodon recognizes UGA (Stop) as Sec • Signal (a stem loop configuration) 3’ to the UGA determine Stop or Sec • Dedicated specific elongation factor recognizes the stem-loop and substitutes for usual elongation factor (EF-Tu)

  8. Degeneracy—Wobble Hypothesis • Explains how some tRNA recognize more than one codons • tRNA molecules only need to make strong base pairs with 2 of the three codons in the nucleotide • This third loose base pairing interaction is called wobble • Note: only certain bases can substitute for others

  9. Wobble Example This UCA codon was read by the tRNA with a UGA anticodon But if this UCA was UCG, it would still have been read by the tRNA with a UGA anticodon

  10. Codon Usage • More than one codon exist for most amino acids (except Met and Trp) • Organism may have a preferred codon for a particular amino acid • Codon usage correlates with abundance of tRNAs (preferred codons are represented by abundant tRNAs) • Rare tRNAs correspond to rarely used codons • mRNAs containing rare codons experience slow translation • Implications for GE?

  11. Amino Acyl Synthetase and tRNA • Amino acyl synthetases catalyze attachment of aa to its appropriate tRNA • One for each amino acid • tRNA • Derived from large 1º transcript • Heavily modified, unusual bases • Extensive folding due to internal H-bonding

  12. Amino Acyl Synthetase • Carboxy end of aa attached to -phospate of ATP • AMP released as carboxy end of amoinoacyl group transferred to O at C-3 of 3’nt • When aa is attached, tRNA is charged or acylated • No aa = uncharged • Wrong aa = mischarged • NOTE PPi

  13. 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: O HO 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

  14. tRNA Function and Structure acceptor stem • Anticodon-complementary to codon on mRNA • Amino attachment (CCA) site • Other recognition sites • DHU loop • TC Loop • Extra arm (variable) • NOTE: also unusual bases observed acceptor stem

  15. tRNA Recognition by Amino Acyl Synthetase • Sequence elements in each tRNA are recognized by its specific synthetase including: • One or more of 3 bases in acceptor stem • Base at position 73 “Discriminator base” • Seems to play a major role in many cases, but in other cases it is completely ignored. • In many, at least one anticodon base

  16. Recognition (cont’d) No common set of rules for tRNA recognition !!! • Anticodon region is not the only recognition site • The "inside of the L" and other regions of the tRNA molecule are also important • Specificity of several aminoacyl-tRNA synthetases determined by: • one or more bases in anticodon • one or more bases in the acceptor stem • discriminator base 73

  17. Mischarging • Observation: several aa similar in size and shape, but mischarging rare. • Editing carried out by aa rRNA synthetase • Ex Double sieve of isoleucine synthetase • Activation site– coarse sieve, rejects aa larger than ile. excluded because they don’t fit. • Editing (hydrolytic) site—fine sieve. Accepts activated amino acids that are smaller than ile (ex, Val-AMP), but rejects Ile-AMP (too large). those that get through are hydrolyzed to aa and AMP. Reduces mischarging from 1/225 (expected) to 1/180,000 (observed). • Sites can also distinguish based on hydrophobicity

  18. 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

  19. 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

  20. Anticodon (recognizes UGU codon, encodes Cys) Experiment (1962) tRNA-ACA Cell-free extract amino acids & enymes tRNA is charged with Cys Protein has Cys RNA template UGUGUGUGUG... Cys-tRNA-ACA Treat w metal catalyst removes thiol groups Charged amino acid is changed chemically Protein has Ala RNA template UGUGUGUGUG... Ala-tRNA-ACA Once an aminoacyl-tRNA has been synthesized the amino acid part makes no contribution to accurate translation of the mRNA.

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