The tRNA i Met is needed to start synthesis of a polypeptide chain

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2. two types of tRNAs exist for the methionine codon tRNAiMet is exclusively used for initiation of a polypeptide chain tRNAMet is exclusively used for elongation of a polypeptide chain The tRNAiMet is needed to start synthesis of a polypeptide chain the methionine in bacterialtRNAi is modified by addition of a formyl group both are charged by the same aminoacyl tRNA synthetase

3. theeukaryotic Initiation Factors (eIFs)eIF3 and eIF6 are required to keep the ribosomal subunits apart after they have finished the synthesis of a protein Ribosomal subunits have to be kept apart before translation initiation

4. ternary complex tRNAiMet is required for the formation of a pre-initiation complex • aternary complex made up of eIF2, GTP and the tRNAiMetthen binds the small ribosomal subunit together with the stabilizing factor eIF1a to form the 43S pre-initiation complex • This event can be regulated through phosphorylation of eIF2 after which it can no longer exchange GDP for the active GTP.

5. eIF4 is instrumental in recruiting an mRNA to the preinitiation complex • next,a protein complex named eIF4 binds the pre-initiation complex as well as the cap of an mRNA. • An associated helicase activity removes any secondary structure in the 5’ region of the transcript...

6. The initiation complex scans the mRNA for the start codon • ...secondary structure unwinding enables unhinderedmovement of the initiation complex along the mRNA to find the start codon, which is often embedded in a Kozak consensus sequence:ACCAUGG • eventually, thetRNAiMet is positioned correctly at the translation start sitetheinitiation factors are then released following GTP hydrolysis • the scanning requires energy from ATP hydrolysis

7. Bring on the big one! • Finally,with the aid of eIF5 and energy from GTP hydrolysis… • the large 60 S ribosomal subunit is recruited, • the tRNAiMet is correctly positioned at the P site, • The eIFs are released and translation begins

8. theP site of the ribosome isthesite where the initiator tRNA (tRNAiMet) is bound only the tRNAi can bind to this site during ribosome assembly this is why the tRNAiMet is so special... The tRNAi is the only tRNA that can bind the "P" position of the ribosome

9. aternary complexoftheelongation factor (EF) 1 alpha, GTPand atRNAaawhose anticodon matches the next mRNA codonis allowed to enter the ribosomal A site Entry of the next amino acid-loaded tRNA into the A site starts the elongation cycle

10. Energy from GTP hydrolysis promotes a confirmational change in the ribosome • upon binding of the ternary complex in the A site,EF1 alpha is released and concurrent GTP hydrolysis leads to aconformational change in the ribosome. • This change brings the amino acids linked to the tRNAs in the P and A site into close proximity

11. ribozyme -> an enzymatically active RNA molecule The ribosome is a ribozyme • the peptidyltransferase activity, which results in peptide bond formation between the amino acids of the neighbouring aminoacyl-tRNAs, depends on the 23S rRNA in the large ribosomal subunit of prokaryotic ribosomes • the 23S rRNA catalyzes the peptide bond formation between the two amino acids without any help from proteins • the equivalent applies to the 28S rRNA in eukaryotic ribosomes

12. EF2 and energy from GTP hydrolysis promotes translocation of the tRNAs into the E and P site respectively • upon peptide bond formation,EF2-bound GTP is recruited and GTP hydrolysis leads to a conformational change in the ribosome,resulting in the translocation of the tRNA moleculesinto the E and P site respectively • a new elongation cycle can begin

13. In the next elongation cycle... • the tRNA in the E site,now rid of its amino acid,is kicked out of the ribosome during the conformational change associated with EF1 alpha release and GTP hydrolysis • the growing polypeptide chain is always covalently linked to one of the tRNAs in the P or A site 1 2 3 1 2 3 • gets recycled

14. once the ribosome encounters a STOP codon in the A site,this codon is recognized by a termination (release) factor,eRF1 eRF1 is thought to structurally mimic aminoacyl-tRNAs another factor, GTP-bound eRF3, then associates with eRF1 UAG RF1 UAA RF2 UGA Translation termination involves termination factors that recognize the stop codons in prokaryotes, two release factors for different STOP codons exist:

15. eRF3 terminates translation by releasing the polypeptide chain • hydrolysis of the GTP bound by eRF3 is necessary for the cleavage of the last amino acid in the P site from its tRNA • this results in the release of the polypeptide chain and the disassembly of the ribosome

16. Interactions between the 5’ and 3’ end of the translated mRNA favour reinitiation • Protein:protein interactions indicate that the complexes associated with the 3’ end of the mRNA interact with protein complexes associated with translational initiation. • Maintaining the 3’ terminal region of the mRNA in close proximity to the site of translational initiation could favour reinitiation. • Efficient translation often is associated with stabilisation