Eukaryotic Translation & it's Regulation.pptx

Eukaryotic Translation & its Regulation

1. WELCOME

2. Assignment PRESENTATION Eukaryotic translation & IT’s regulation

3. What are Eukaryotes ? Eukaryotes are organisms with a complex cell or cells, in which the genetic material is organised into a membrane bound nucleus or nuclei and it also contains cell orgenelles .

4. What are Translation ? Translation is basically a synonym process of protein synthesis. Translation is the process by which protein is synthesized from the information contained in a molecule of messenger RNA (mRNA). It can defined as “the process by which the sequence of nucleotides in a messenger RNA molecule directs the incorporation of amino acid into protein”.

5. Translation : An Overview Ribosomes translate the genetic message of mRNA into proteins. The mRNA is translated from 5` 3`. Amino acids bound to tRNA are inserted in a proper sequence due to : Specific binding of each amino acid to its tRNA . Specific base-pairing between the mRNA codon and tRNA anticodon .

6. Components of Translation mRNA : - Made in the nucleus, transported to cytoplasm. tRNA : - Adaptor molecule that mediate the transfer of information form nucleic acid to protein. Ribosomes : - Manufacturing units of a cell. Enzymes : - Required for the attachment of amino acids to the correct tRNA molecule, and for peptide bond formation between amino acids. Proteins : - Soluble factors necessary for proper initiation, elongation and termination.

7. Enzymes Aminoacyle-tRNA Synthetases catalyze the attachment of tRNA molecule to its respective amino acid. - At least one for each tRNA . - Attachment of amino acid activates/changes the tRNA molecule. Peptidyl Transferase - Forms the peptide bond between the amino acids.

8. TRANSLATION MACHINERY The machinery required for translating the language of messenger RNAs into the language of proteins is composed of four primary components. mRNA : Messenger RNA (mRNA) provides an intermediate that carries the copy of a DNA sequence that represents protein. tRNA : tRNA acts as an adaptor between the codons and the amino acids they specify. Enzymes : Required for the attachments of amino acids to the correct tRNA molecule. Aminoacyle-tRNA Synthetases . - Attachment of amino acid charges the tRNA molecule. b. Peptidyl Transferase . - Forms the peptide bond between the amino acids. Ribosome : It is the macromolecular complex that directs the synthesis of proteins.

9. TRANSLATION PROCESS In a prokaryotic cell, transcription and translation are coupled; that is, translation begins while the mRNA is still being synthesized. In a eukaryotic cell, transcription occures in the nucleus, and translation occurs in the cytoplasm. Translation involves three major steps : INITIATION ELONGATION TERMINATION

10. 1. INITIATION The initiation of translation in eukaryotes is complex, involving at least 10 eukaryotic initiation factors ( eIFs ) & divided into 4 steps : Ribosomal dissociation. Formation of 43S preinitition complex. Formation of 48S initiation complex. Formation of 80S initiation complex.

11. a. Ribosomal dissociation The 80S ribosome dissociates to form 40S & 60S subunits. Two initiating factors namely eIF-3 & eIF-1A bind to the newly formed 40S subunit & thereby block its reassociation with 60S subunit.

12. b. Formation of 43S preinitition complex. A ternery complex containing met- tRNA ’ & eIF-2 bound to GTP attaches to 40S ribosomal subunit to form 43S preinitiation complex. The presence of eIF-3 & eIF-1A stabilizes this complex.

13. c. Formation of 48S initiation complex. The binding of mRNA to 43S preinitiation complex results in the formation of 48S initiation complex through the intermediate 43S initiation complex. eIF-4F complex is formed by the association of eIF-4G, eIF-4A with eIF-4E. The eIF-4F (referred to as cap binding protein) binds to the cap of mRNA.

14. Then eIF-4A & eIF-4B bind to mRNA & reduce its complex stucture . This mRNA is then transferred to 43S complex. For the appropriate association of 43S preinitiation complex with mRNA, energy has to be supplied by ATP. The ribosomal initiation complex scans the mRNA for the identification of appropriate initiation codon . 5’-AUG is the initiation codon .

15. d. Formation of 80S initiation complex. 48S initiation complex binds to 60S ribosomal subunit to form 80S initiation complex. The binding involves the hydrolysis of GTP (bound to eIF-2). This step is facillatated by the involvement of eIF-5. As the 80S complex is formed, the initiation factors bound to 48S initiation complex are released & recycled.

16. 2. ELONGATION Ribosomes elongate the polypeptide chain by a sequential addition of amino acids. The amino acids sequence is determined by the order of the codons in the specific mRNA. Elongation, a cyclic process involving certain elongation factors ( Efs ). Elongation may be divided into three steps. Binding of Aminoacyl t-RNA to A-site. Peptide bond formation. Translocation.

17. a. Binding of Aminoacyl t-RNA to A-site The 80S initiation complex contains met tRNA in the P-site & A-site is free. Another Aminoacyl-tRNA is placed in the A-site. This requires proper codon recognization on the mRNA & involvement of elongation factor 1a(EF-1a) & supply of energy by GTP. The aminoacyl t-RNA is placed in the A-site,EF-1a & GDP are recycled to bring another Aminoacyl-tRNA .

18. b. Peptide bond formation The enzyme Peptide transferase catalyzes the formation of peptide bond. The activity of this enzyme lies on 28S RNA of 60S ribosomal subunit. It is therefore the rRNA (and not protein) referred to as ribozyme that catalyzes peptide bond formation. Net result of peptide bond formation is the attachment of the growing peptide chain to the tRNA in the A-site.

19. c. Translocation The ribosome moves to the next codon of the mRNA. This process called translocation, involves the movement of growing peptide chain from A-site to P-site. Translocation requires EF-2 & GTP. GTP gets hydrolyzed and supplies energy to move mRNA. EF-2 & GTP complex recycles for translocation. About six amino acids per second are incorporated during the course of elongation of translation in eukaryotes.

20. Step 1 : Decoding Elongation Cycle Step 2 : Peptide bond formation Step 3 : Translocation

21. 3. TERMINATION One of the stop or termination signals(UAA, UAG and UGA) terminates the growing polypeptide. When the ribosome encounters a stop codon , - there is no tRNA available to bind to the A site of the ribosome, - instead a release factor binds to it. In eukaryotes, a single release factor-eukaryotic release factor 1 (eRF-1)-recognizes all three stop codons , and eRF-3 stimulates the termination events. Once the release factor binds, the ribosomes unit falls apart, - releasing the large and small subunits, - the tRNA carrying the polypeptide is also released, freeing up the polypeptide product. Ribosome recycling occures in eukaryotes.

22. Regulation by RNA binding protein Inhibitory effect on translation except PABP. For translation these protein must be degraded.

23. Regulation by specific 5’ UTR protein interaction : Rare but just one example : ferritin mRNA (iron). For strong inhibition of translation requires the protein RNA interaction at cap proximal location, prevent loading of 43S complex onto mRNA. Inhibition is much weaker if protein binding RNA motif moved to more cap distal position. In this 43S complex loaded over the mRNA, it’s subsequent scanning will displace the bound protein.

24. Stimulation by 3’ poly tail : PABP have stimulatory effect. PABPs second RRM domain interact with eIF4G, result in the circulation of mRNA in the closed loop configuration. By the help of PABP eIF4F remain anchor to mRNA at 3’ poly A tail, this anchoring will not be occure in the absense of PABP or poly A tail.

25. Regulation by specific 3’ UTR protein interaction : Control by 3’UTR is entirely dependent on changes in poly A tail length, because the regulatory mRNA are repressed when they have a short tail or activated when they have a long length. In some cases translation can be activated with out the lengthening of short poly A tail, in some cases protein may changes the polyadenylation status.

26. Translation regulation by miRNAs : Sequence specific. Repress translation at 3’UTR Can act in conjunction with RNA binding protein Almost 21 nucleotide Degree of repression increases with the increasing number of miRNA Repression efficiency might also be influenced by the distance and sequence between miRNA target sites and also their position in the 3’UTR In some cases miRNA act as a adaptor for sequence specific RNA binding protein.

27. The mechanism of repression have 2 main component - Normal mRNA translation - Deadenylation dependent pathway Repressed mRNA displace from large polysome to small polysome or sub polysomal partical , this indicate the inhibition of initiation.

28. Phosphorylation of eukaryotic translation initiation factor eIF4E contributes to its transformation and mRNA transport activities The eukaryotic translation initiation factor eIF4E is dysregulated in a wide variety of human cancers. In the cytoplasm, eIF4E acts in the rate-limiting step of translation initiation whereas in the nucleus, eIF4E forms under nuclear bodies. Thus, phosphorylation of nuclear eIF4E seems to be an important step in control of the mRNA transport and thus the transforming properties of eIF4E. Topisirovic et al. (2004)

29. Controlling gene expression through RNA regulations : The role of the eukaryotic translation initiation factors eIF4E The eukaryotic translation initiation factor eIF4E is a potent oncogene . In fact its overexpression in human cancer often correlates with poor progenosis . Additionally, eIF4E may play a role in mRNA sequestration and stability in cytoplasmic processing bodies. Hence, eIF4E functions as a central node of an RNA regulation, which plays an essential role in normal differentiation and development and is frequently dysregulated in cancer. Here, the physiological implications of these observations are described and the clinical implications of directly targeting eIF4E, and related regulon are discussed. Culikovic et al. (2007)

30. Source: Genome 4 by T. A. Brown Topisirovic I. (2004). Phosphorylation of eukaryotic translation initiation factor eIF4E contributes to its transformation and mRNA transport activities. 64 : 8639-8642. Culjkovic B. (2007). Controlling gene expression through RNA regulations : The role of the eukaryotic translation initiation factors eIF4E. 6: 65-69.

31. Thank you