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TRANSLATION

BIOCHEMISTRY

mprasadnaidu
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TRANSLATION

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  1. TRANSLATION M.Prasad Naidu MSc Medical Biochemistry, Ph.D,.

  2. THE LANGUAGE OF NUCLEOTIDE SEQUENCEON m-RNA ( GENTIC CODE ) IS TRANSLATED IN TO THE LANGUAGE OF AMINIO ACID SEQUENCE.

  3. t-RNA ( soluble RNA) Transfer A.A from cytosol to the ribosomal protein synthesising machinery. 73 – 93 nucleotides in length

  4. RIBOSOMES INFRASTRUCTURE FOR THE m-RNA, t-RNA & A.A TO INTERACT WITH EACH OTHER FOR TRANSLATION. 80S IN EUKARYOTES & 60S IN PROKARYOTES. LARGER 60S (50S) SUBUNIT SMALLER 40S (30S) SUBUNIT

  5. Requirements for TRANSLATION: ribosomes m-RNA t-RNA A.As AMINOACYL t-RNA SYNTHETASES ATP, GTP ENZYMES OF TRANSLATION PROTEIN FACTORS

  6. TRANSLATION: Steps 1. ACTIVATION OF A.A 2.INITIATION 3.ELONGATION 4.TERMINATION 5.POST TRANSLATIONAL PROCESSING

  7. ACTIVATION OF A.A • AMINOACYL t-RNA SYNTHETASE • A.A + t-RNA + ATP  Aminoacyl t-RNA + AMP

  8. ACTIVATION OF A.A AMINOACYL t-RNA SYNTHETASE D ARM OF tRNA RECOGNISED BY THE ENZYME A.A + t-RNA + ATP  Aminoacyl t-RNA + AMP ENZYME IDENTIFIES THE SPECIFIC t-RNA TRANSFERS AMINOACYL GROUP TO 3’ END CCA -COOH GROUP OF A.A ESTERIFIED WITH THE 3’ OH GROUP OF t-RNA. AT LEAST 1 ENZYME FOR 1 A.A

  9. mRNA is read in 5’ to 3’ direction • PROTEIN SYNTHESIS TAKES PLACE FROMN-TERMINAL TO C-TERMENAL END

  10. INITIATION: • Basic requirements for initiation • Ribosome • mRNA to be translated • The initiating Met-tRNA • A set of protein factors called initiation factors. • In Eukaryotes there are at least nine factors, designated • e If to indicate eukaryotic origin. • In prokaryotes, three initiation factors are known IF1, IF2 and IF3

  11. Steps involved in Initiation: 1. Ribosomal dissociation 2. Formation of 43S pre-initiation complex 3. Formation of 48S initiation complex 4. Formation of 80S initiation complex

  12. Ribosomal dissociation: • eIF3 and eIF1 A binds to 40S subunit and prevents the reassociation with 60S – causing ribosomal subunit dissociation.

  13. 2. Formation of 43S pre-initiation complex GTP + eIF2 This complex binds to met-tRNA-I This GTP-eIF2-tRNA-I complex binds to the 40S ribosomal subunit to form 43S pre-initiation complex.

  14. 3. Formation of 48S initiation complex Binding of m-RNA to 43S pre-initiation complex forms 48S initiation complex. 5’ end of m-RNA contains CAP which helps in binding of m-RNA to 43S pre-initiation complex. The association of mRNA with 43S initiation complex requires: CAP binding protein, eIF4, ATP KOZAK CONSENSUS (EUKARYOTES) on mRNA facilitates binding of mRNA to pre-initiation complex SHINE DALGARNO SEQUENCE on mRNA facilitates binding of mRNA to pre-initiation complex

  15. 4. FORMATION OF 80S RIBOSOME ASSEMBLY 48S INITIATION COMPLEX + 60S RIBOSOME. + e-IF2 + e-IF5. e-IF 5 HAS GTPase ACTIVITY. GTP BOUND IN STEP 1 HYDROLYSED FOR ENERGY. ALL INITIATION FACTORS NOW RELEASED. NEXT STAGE IS ELONGATION.

  16. 80S ribosome Ribosomal dissociation 40S subunit Ternary complex

  17. 43S Preinitiation complex eIF eIF4 eIF Cap Initiating codon 48S Inititation complex 60S eIF 80S Inititation complex

  18. P & A SITES RIBOSOME CONTAINS 2 RECEPTOR SITES FOR t-RNA MOLECULES P (PEPTIDYL) SITE A (AMINOACYL) SITE Met-t-RNA OCCUPIES P SITE. ALL OTHER t-RNAs OCCUPY A SITE t-RNA BINDS WITH RIBOSOME THROUGH THE PSEUDOURIDINE ARM ANTICODON OF t-RNA & CODON ON m-RNA BIND A SITE IS FREE NOW.

  19. ELONGATION: BINDING OF NEW AMINOACYL t-RNA CODON IN m-RNA DETERMINES THE INCOMING A.A EF 1 & GTP REQD. PEPTIDE BOND FORMATION: -NH2 GROUP OF INCOMING A.A IN A SITE FORMS PEPTIDE BOND WITH COOH GROUP OF A.A INP SITE. ENZYME IS PEPTIDYL TRANSFERASE NO NEED FOR ENERGY AS A.A IS ACTIVATED.

  20. TRANSLOCATION: WHEN PEPTIDE BOND FORMED Met FROM P SITE SHIFTED TO A SITE. t RNA IS RELEASED FROM P SITE, NOW P SITE FREE RIBOSOME MOVES FORWARD BY 1 CODON PEPTIDYL t-RNA TRANSLOCATED TO P SITE.NOW A SITE EMPTY NEW AMINOACYL t-RNA WILL COME ONLY TO A SITE. STEP REQUIRES EF 2 AND ENERGY FROM GTP. NEW AMINOACYL t-RNA CAN COME IN ELONGATION CONTINUES….

  21. P A P P A A P A P A P A

  22. ENERGY REQUIREMENTS: ACTIVATION OF A.A 2 ATP FOR PRE-INITIATION COMPLEX 1 GTP FOR INITIATION COMPLEX 1 ATP BINDING OF AMINOACYL t-RNA 1 GTP TANSLOCATION PROCESS 1 GTP

  23. TERMINATION; SUCCESSIVE ADDITIONS OF A.A TERMINATION CODONS REACHED. NO t-RNA CORRESPONDING TO TERMINATION CODON A SITE REMAINS FREE RELEASING FACTOR ENTERS A SITE RELEASES THE PEPTIDE CHAIN WITH HYDROLYSIS OF GTP. RIBOSOME DISSOCIATES INTO 60 AND 40S SUBUNITS.

  24. A P A P A P

  25. POLYRIBOSOME (POLYSOME)

  26. PROTEIN TARGETTING: PROTEINS FOR EXTERNAL SECRETION: SYNTHESISED ON RER SIGNAL PEPTIDE (LEADER sequence) THRO ER, GOLGI REACHES CORRECT DESTINATION. INTERNAL PROTEINS: LACK SIGNAL PEPTIDE FREE RIBOSOMES DO NOT ENTER ER. ARE AVAILLABLE IN CYTOPLASM.

  27. THANK YOU

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