TRANSLATION & POST TRANSLATION MODIFICATIONS.pptx

Translation and post translational modification

1. TRANSLATION & POST TRANSLATIONAL MODIFICATIONS DR.K.MAGILA,MD(BIOCHEMISTRY)

2. CODING & NON-CODING RNA CODING RNA mRNA NON-CODING RNA SnRNA , miRNA,rRNA , tRNA

3. DNA RNA Protein Transcription Translation 1 ) mRNA 2) rRNA 3) snRNA 4) tRNA RNA FORMATION Carried out by RNA POLYMERASE mRNA is the only type of RNA that is translated into protein

4. COMPONENTS REQUIRED FOR TRANSLATION

5. COMPONENTS REQUIRED FOR TRANSLATION 1 . Aminoacids 2. tRNA 3. mRNA 4. rRNA 5. Ribosomes 6. Enzymes: Aminoacid tRNA synthetase Peptidyl transferase 7. eukaryotic initiation factors – elf s 8. Energy source: ATP ,GTP

6. Transfer RNA It varies in length from 73 to 93 nucleotides . Function: They transfer the aminoacids from cytoplasm to protein synthesizing machinery, hence called t RNA.

7. Transfer RNA They are easily soluble, hence called soluble RNA. There are atleast 20 tRNA one corresponding to each of the 20 aminoacids required for protein synthesis.

8. t RNA – Clover leaf structure It contains 5 main arms . a) Acceptor arm b) Anticodon arm c) D HU arm d) T ψ C arm e) Extra arm

9. Secondary structure – t RNA Acceptor arm The acceptor arm is at 3’ end It has 7 base pairs The end sequence is unpaired Adenine,Cytosine,Cytosine at the 3’ end CCA occurs postrancriptionally . The t-RNA appropriate aminoacids is attached onto the 3’ – OH group of the a moiety of the acceptor arm .

10. Secondary structure – t RNA Anticodon arm : Lies at the opposite end of the acceptor arm 5 base pairs long Recognizes the triplet codon and binds to the complementary codons present in the m RNA.

11. Anticodon arm : Specificity of t RNA lies in the anticodon arm. Example : if m RNA has a codon with the sequence of UUU Anticodon sequence will be AAA UUU codes for phenylalanine

12. Anticodon arm : UUU AAA t RNA is called adapter molecule

13. Secondary structure – t RNA DHU arm It has 3-4 base pairs. Serves as the recognition site for the enzyme ( amino acyl t RNA Synthetase ) that adds aminoacid to the acceptor arm. T ψ C arm This is opposite to DHU arm It is involved in the binding of t RNA to the ribosomes

14. Secondary structure – t RNA Extra arm or variable arm . About 75 % of t RNA possess a short extra arm. If about 3 – 5 base pairs present , t RNA belongs to class 1 . Class 2 t RNA have 13 to 21 base pairs in length. Majority t RNA belongs to class 2.

15. PROCESSING OF t RNA RNA polymerase III transcribes t RNA. Precursor t RNA is trimmed at 5’ and 3’ end to form mature t RNA. 5’ end is trimmed by an enzyme called RNAse P ( Ribozyme ) 3’ end – Addition of CCA residues

16. RIBOSOMAL RNA Ribosomes provides necessary infrastructure for aminoacids , t RNA, mRNA.

17. Ribosomal RNA – (r RNA ) Human ribosomes - larger one (60S ) and a smaller subunit(40S) S = Svedberg units , a parameter sensitive to molecular size & shape. 60S subunit = 5.8S r RNA, & 28SRNA ( peptidyl tranferase role) 40S Subunit has 18 s r RNA

18. FORMATION OF r RNA Formed as a large single precursor molecule. Then it is cleaved into three final products- 5.8S r RNA, & 28SRNA ,18 s r RNA

19. TRANSLATION

20. TRANSLATION Translation is a cytoplasmic process . The mRNA synthesized in the nucleus is transported to the cytoplasm, where the mRNA is translated from 5’ to 3’ end . In the polypeptide chain synthesized, the first amino acid is the amino terminal one. The chain growth is from amino terminal to carboxyl terminal.

21. Activation of amino acid Initiation Elongation Termination and Post-translational processing. Translation Process STEPS OF TRANSLATION

22. There is at least one tRNA for each of the 20 amino acids. The D arm of tRNA is very important for the recognition by the enzyme. CCA 3’ terminus of the acceptor arm carries amino acid. Aminoacyl tRNA synthetase Amino acid + tRNA + ATP --------------  Aminoacyl tRNA+ AMP Activation of Amino Acid (Charging Reaction) ACTIVATION OF AMINO ACID

23. ACTIVATION OF AMINO ACID

24. INITIATION OF PROTEIN SYNTHESIS 1 : RECOGNITION STEP 1 st Aminoacid incorporated is methionine ( AUG codon) – Eukaryotes 1 st Aminoacid incorporated is N-Formyl methionine – Prokaryotes

25. STEPS of INITIATION 1. Recognition step 2. met-tRNA, GTP, elf -2 to 40s Ribosome to form Pre Initiation Complex 3. Binding of m RNA with the help of poly A tail to 40 s PIC - 43s IC 4. 43 s IC + 60 s ribosomal unit - 80 s IC

26. RECOGNITION STEP KOZAK sequence in eukaryotes

27. (UAC: anticodon on Met-tRNA; AUG: start signal; P: peptidyl site; A: aminoacyl site). INITIATION 43S Preinitiation Preinitiation complex

28. 60S RIBOSOMAL UNIT P: peptidyl site : Growing polypeptide chain A: aminoacyl site): new incomimg tRNA is added E site ( exit site ) : Deacylated tRNA

29. ELONGATION PROCESS OF TRANSLATION Binding of aminoacyl tRNA to A site . Peptide bond formation Translocation of ribosome on the mRNA

30. ELONGATION

31. TERMINATION OF TRANSLATION . RF- Releasing factor 1,3

32. For each peptide bond formation, four high energy phosphate bonds are used. Two for the initial activation and one for EF-1 step (GTP to GDP), and one for EF-2 step (GTP to GDP). Actual peptide bond formation (peptidyl transferase step) does not require any energy, because the amino acids are already activated. Further, 1 ATP is used for initiation complex formation; 1 GTP for 80S ribosome formation and 1 GTP for termination. Energy Requirements ENERGY REQUIREMENYTS

33. POLYRIBOSOME During protein synthesis , many ribosomes can be associated with any m RNA ,molecule to form an assembly called the polysome

34. Proteolytic cleavage , eg , pro-insulin to insulin Modification of amino acids 2-a Gamma carboxylation of glutamic acid of prothrombin (vitamin K dependent) 2-b Hydoxylation of proline and lysine in collagen (vitamin C dependent) Subunit aggregation, eg . Hemoglobin, Immunoglobulin Post-translational Processing POST TRANSLATIONAL MODIFICATIONS

35. Post-translational processing of insulin by proteolytic cleavage.

36. Phosphorylation Eg : Serine, threonine residues undergoes phosphorylation in Glycogen phosphorylase. Glycosylation : O- glycosidic linkage through s erine,threonine . N- glycosidic linkage through asparagine & glutamine Methylation & Acetylation of histones

37. Reversible Irreversible Disulfide bridge Glycosylation Phosphorylation N-acetylation ADP-ribosylation   Proteolysis Ubiquitination Lysine hydroxylation Methylation   Post-translational Modifications

38. When a protein is being synthesized, it may assume different three-dimensional structures, out of which only one will have the biological activity. Abnormal folding of proteins may lead to protein misfolding diseases . Chaperones help to produce the correct spatial arrangement. Chaperones attach to nascent polypeptide chains and prevent wrong foldings ; so that the folding is allowed only in the correct direction. Chaperonopathies are disorders resulting from “sick chaperones”. These diseases progress with age. Protein Folding and Chaperones PROTEIN FOLDING

39. Over polyribosomes rough endoplasmic reticulum is present

40. Zellweger syndrome is due to defective oxidation of very long chain fatty acids (VLCFA. Peroxisomal enzymes are produced; but their entry into peroxisome is denied. This leads to insufficient oxidation of VLCFA. Accumulation of VLCFA in CNS causes neurological impairment and death in childhood. Defects in Protein Targeting

41. P rimary hyperoxaluria , which causes kidney stones at an early age. The defect is due to protein targeting defect . enzyme alanine glyoxalate aminotransferase is seen in mitochondria, instead of its normal peroxisomal location. Familial hypercholesterolemia is due to deficient transport signals. Inclusion cell disease is due to non-entry of normal enzymes into lysosomes. Mannose-6-phosphate which is the marker to target enzymes to lysosomes; is absent in this disease.

42. INHIBITORS OF TRANSLATION

43. They generally act only on bacteria and are nontoxic to human beings. This is because mammalian cells have 80S ribosomes, while bacteria have 70S ribosomes. Reversible Inhibitors in Bacteria These antibiotics are bacteriostatic. Tetracyclins inhibit attachment of aminoacyl tRNA to the A site of ribosomes. Chloramphenicol inhibits the peptidyl transferase activity of bacteria. Erythromycin and clindamycin prevent the translocation process. Irreversible Inhibitors in Bacteria These antibiotics are bactericidal. Streptomycin binds to 30S subunit of bacterial ribosomes. They cause misreading of mRNA and totally inhibit protein synthesis. Inhibitors of Protein Synthesis

44. Inhibitors of Protein Synthesis in Eukaryotes Puromycin is structurally similar to tyrosine-tRNA and gets attached to the "A" site of the ribosome. So, the incomplete peptide is released. Cycloheximide inhibits peptidyl transferase in 60S subunit. Diphtheria toxin, liberated by the bacteria, Corynebacterium diphtheria, causes inactivation of EF-2 by attachment of ADP to EF-2 and consequent inhibition of protein biosynthesis in mammalian systems. Inhibitors of Protein Synthesis

45. Differences between Prokaryotic and Eukaryotic Translation Feature Prokaryotes Eukaryotes Ribosomes 30S small subunit and 50S large subunit associate to form 70S ribosome 40S small subunit and 60S large subunit together form the 80S ribosome Initiation Translation starts before transcription is completed. No mRNA processing Transcription and translation are well separated and the processed mRNA is translated Consensus sequences Initiating codon AUG is recognised by the Shine Dalgarno sequence Initiating codon AUG is recognised by Kozak sequence   Initiator tRNA The initiating tRNA carries formylated methionine The initiating tRNA carries methionine

46. Differences between Prokaryotic and Eukaryotic Translation Feature Prokaryotes Eukaryotes Formation of initiation complex Initiation factors and GTP hydrolysis are required for the binding of the small ribosomal subunit to the mRNA Several IF s and GTP hydrolysis are required for the formation of the initiation complex. Peptide bond formation The peptidyl transferase ribozyme is in the 23S r RNA Peptidyl transferase activity in the 28S rRNA of the large subunit. Post-translational events Mainly co-translational modifications Mainly post-translational modification, sorting, export and localisation Inhibition Antibiotics can inhibit different stages of translation   Toxins like diphtheria, ricin, puromycin and cycloheximide

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