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A9 Translation

A9 Translation. Role of transfer RNA Codon recognition Translation Initiation Elongation Termination Post-translational modifications. 1.Role of transfer RNA.

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A9 Translation

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  1. A9 Translation

  2. Role of transfer RNA • Codon recognition • Translation • Initiation • Elongation • Termination • Post-translational modifications

  3. 1.Role of transfer RNA • Translation: the process by which cell syn-thesize proteins, in which the information encoded in mRNA molecules is used to specify the amino acid sequence of a protein. • Role of transfer RNA: deliver amino acids to the ribosome in an order specified by the mRNA sequence.

  4. Isoacceptors: tRNAs that bind the same AA • Aminoacylation: also called charging. The AA covalently attached to the end of the acceptor arm of the tRNA, catalyzed by aminoacyltRNAsynthetases with the hydrolysis of ATP. Each enzyme can charge all the isoacceptortRNAs for that AA.

  5. 2. Codon recognition • The anticodon in the anticodon loop of tRNA binds to the codon by complementary base-pairing • Degeneracy of the genetic code: more than one codon represent one AA

  6. Third base degeneracy or Wobble: the third base of codon are not perfectly base-paired with the anticodon, e.g. G:U,C; I:C,A,U, • Wobble allows a single tRNA to decode more than one member of a codon family decreasing the number of tRNAs required by the cell, serves to minimize the effects of mutations

  7. 3. Translation • Mechanism similar in prokaryotes and eukaryotes, including three stages: Initiation, Elongation and Termination • Sets of accessory proteins assist ribosome • Energy is provided by GTP (for ribosome movement and accessory binding) and ATP (charging tRNAs, removing secondary structure from mRNA)

  8. 4. Initiation • Binding of small ribosomal subunit to the mRNA • In prokaryotes, at the Shine-Dalgarno sequence (5’AGGAGGU3’), then migrates in a 3’ direction along the mRNA until it finds the AUG

  9. In eukaryotes, recognize the cap structure of mRNA, then moves downstream until it encounters the first AUG

  10. tRNA charged with methionine binds to the AUG: • In bacteria, the methionine is modified by addition of a formyl group (-CHO) to one of the hydrogens of the amino group, the combination of mRNA, small ribosomal subunit and the tRNAfmet is called the initiation complex • 在原核生物中，甲酰化的甲硫氨酸tRNA 参与起始 • In eukaryotes, the methionone is not formylated • 真核生物的甲硫氨酸tRNA 不需要甲酰化直接参与

  11. Initiation factors: • In prokaryotes, IF1 and IF3 bind to small ribosomal subunit first, followed by IF2 complexed with GTP→The small ribosomal subunit then binds the mRNA and locates the AUG initiation codon→the initiator tRNA binding and IF3 is released→IF1 and IF2 are released once complete ribosome forms

  12. In eukaryotes, more initiation factors.eIF2 and eIF3 have roles similar to bacterial IF2 and IF3. Several of the eukaryotic initiation factors are involved in removing secondary structure from mRNA before it is translated.

  13. Translation initiation codon: • In prokaryotes, AUG,GUG or UUG • In eukaryotes, AUG

  14. The initiation of translation in bacterial cells requires several initiation factors and GTP.

  15. 5.Elongation • Large ribosomal subunit binding to form complete ribosome: • P(peptidyl) site: the site for tRNAfmet and peptide • A (aminoacyl )site: site for other entered tRNA charging AA • A tRNA enters the A site and base pairs with the second codon, elongation begins

  16. Peptide bond forms between the carboxyl group of methionine and the amino group of the second AA, catalyzed by peptidyl transferase. tRNA deacylase breaks the link between methionine and its tRNA after the formation of peptide bond

  17. In prokaryotes, EF-Tu is associated with entry of a tRNA into the A site. EF-Tu/GTP→GTP hydrolyzed→EF-Tu/GDP→EF-Tu/EF-Ts→EF-Tu/GTP (EF-Tu/EF-Ts cycle) • In eukaryotes, eEF-1 brings the tRNA to the A site, the reaction is also associated with GTP

  18. Ribosome moves on to the next codon: the newly formed peptide moves into the P site expelling the uncharged tRNA and the A site becomes vacant, a third charged tRNA enters A site and the elongation cycle is repeated. The ribosome translocates to the next codon.

  19. The elongation oftranslation comprises three steps

  20. In prokaryotes, EF-G binds to the ribosome in a complex with GTP which is hydrolyzed providing energy for translocation • In eukaryotes, eEF-2 is equivalent to EF-G

  21. Polysomes: mRNAs are translated by several ribosomes at once forming a polysome （a）兔网织红细胞；（b）唾液腺细胞的电镜照片

  22. 6. Termination • Termination codon enters the A site and are recognized by release factors and cause the completed polypeptide to be released.

  23. In E coli, RF1 and RF2 recognize UAA, UAG and UAA, UGA respectively, RF3 plays an ancillary role in this process • In eukaryotes, eRF, with GTP hydrolysis, end translation

  24. Translation ends when a stop codon is encountered. Conclusion: When a stop codon is encountered, release factors associate with the ribosome and bring about the termination of translation.

  25. 7. Post-translational modifications • Covalent attachment of chemical groups and cleavage of the polypeptide chain: • Addition of small groups: methylation,phos-phorylation, acetylation,hydroxylation

  26. Addition of larger molecules: lipids, oligo-saccharides • Trimming of AAs: removal of internal peptides, removal of amino-terminal signal peptide sequence et al.

  27. Questions • Explain wobble and its role. • Describe the process of translation, find the difference between prokaryotes and eukaryotes • Post-translational modifications and their roles.

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