Translation

Translation

1. TRANSLATION By- Sanju Sah St. Xavier’s college, maitighar , Kathmandu Department of Microbiology 1

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3. Introduction Translation - conversion of mRNA base sequences into protein amino acid sequences which involves a step wise reading of the message present in mRNA base sequences as codons and translating it into the amino acid sequences of protein. The translation of mRNA messages requires the following two RNA species. Transfer RNA( tRNA ) Ribosomal RNA( rRNA ) 3

4. Different RNAs 1. Messenger RNA(mRNA) Messenger RNA is a single stranded base-for-base complementary copy of one DNA strand of the coding sequence of a gene and provides the information for amino acid sequence of the polypeptide specified by that gene. It functions as a template for synthesis of protein. Least stable type of RNA. 5-10% of the total cellular RNA. In Eukaryotes mRNA has certain modification after its synthesis, considered as post transcriptional modification. 4

5. Polycistronic and monocistronic mRNA Generally, a single prokaryotic mRNA codes for more than one poylpeptide , such an mRNA is called as polycistronic mRNA. Some correspond to a single gene known as monocistronic mRNA. All eukaryotic mRNAs are monocistronic . It contains three regions: coding region, 5’ leader region and 3’ trailer region. The coding region usually begins with AUG codon and ends with a termination codons, the codons in between coding for the different amino acids of the concerned polypeptide. The leader sequence is present at the 5’end before the AUG codon and is not translated. The trailer sequence is located beyond the termination codon of the last coding region which is also not translated. 5

6. 2. Transfer RNA ( tRNA ) Comprises about 10-15% of total cellular RNA. tRNA is transcribed in nucleus and must enter the cytoplasm. Each tRNA molecule links to a particular mRNA codon with a particular amino acid. Size of the tRNA varies from 74-95 nucleotides. All tRNA molecules have a proportion of unusual bases; dihydrouridine ( Dih U), pseudouridine , inosine (I), methylated bases and thymine(T). 6

7. Transfer RNA: adaptor between mRNA and amino acids anticodon codon 7

8. Sequence of tRNA ala determined by Robert Holley in 1965. Deduced “cloverleaf” structure 8

9. Common features 1. All single-stranded nucleotide chains of 73-93 ribonucleotides 2. Contain many unusual bases 3. The 5’ end is always phosphorylated and usually a pG 4. The 3’ end is always a -CCA-OH 5. Extensive base pairing, highly conserved 3-D structure Over 100 tRNA molecules have been sequenced 9

10. 6. Anticodon loop contains 7 bases: pyr - pyr - X-Y-Z - modified purine-variable Out of these 7 unpaired nucleotides the middle three form anticodon. Anticodon recognizes and codon of mRNA and binds to it . Over 100 tRNA molecules have been sequenced 10

11. Alexander Rich and Aaron Klug determined the 3-D structure of yeast Phe-tRNA by X-Ray crystallography in 1974 3-D Structure of tRNAs 11

12. 3. Ribosomal RNA( rRNA ) Ribosomal RNA( rRNA ) occurs in association with proteins called ribosomes where amino acids are joined to form protein primary structure - polypeptide Ribosomes of eukaryotes and prokaryotes differ in size and other details. eukaryotic ribosomes - smaller 40S subunit has a single 18S rRNA (1874 bases) and larger 60S subunit has one molecule each of 28S (4718 bases), 5.8S (160 bases) and 5S (120 bases) rRNA . The two subunits of ribosome dissociate in low magnesium (Mg⁺⁺) concentration and re-associate in high Mg⁺⁺ concentration. 12

13. Furthermore, each ribosome dissociates into the two subunits every time it finishes reading an mRNA molecule. The two subunits reunite, at random, to produce complete ribosomes once the smaller subunit attaches itself to he initiation site of an mRNA. The larger subunit of ribosome carries the newly synthesized protein, while the smaller subunit is associated with the mRNA. 13

14. Ribosome Made of protein and rRNA has a large and small subunit has three binding sites for tRNA on its surface has one binding site for mRNA Facilitates codon and anticodon bonding Components of ribosome are made in the nucleus and exported to the cytoplasm where they join to form one functional unit. 14

15. The three tRNA binding sited are: A site: holds tRNA that is carrying the next amino acid to be added P site: holds tRNA that is carrying the growing polypeptide chain E site: where discharged tRNA leave the ribosome. 15

16. The Ribosome Prokaryotic Eukaryotic 30S and 50S subunit 40S and 60S subunit 16

17. The Prokaryotic Ribosome 17

18. 18 The Eukaryotic Ribosome

19. Genetic Code Genetic code - a language that correspond between a sequence of triplet nucleotide bases of mRNA and the sequence of amino acid they specify. Each individual word is composed of three nucleotide bases which are referred as codons. 19

20. Information in DNA to Protein The DNA is first transcribed into mRNA inside the nucleus. mRNA then moves outside the nucleus to the ribosomes ( rRNA ), where protein synthesis takes place. Ribosomes move along the mRNA strand, reading three nucleotides at a time. The genetic code is also read by  transfer RNAs ( tRNA ) . Each tRNA has an anticodon that is complementary to the codon in the mRNA which then translate the codon into aminoacid . due to wobble base pairing  the cell manages with less tRNAs than would be expected from the number of anticodons required to match the codons in the code table. Each tRNA carry the amino acid specified by the codon and joins the amino acids together to make a polypeptide, which later folds into protein . 20

21. features of genetic code Triplet nature The four nucleotide bases ( A,G,C and U) in mRNA are used to produce the three base codons. 64 combinations There are 64 codons in total including 61 sense codons (codons that specify amino acid) and 3 non sense/ stop codons (codon that do not specify amino acid/signal termination). There are 61 codons that code for the 20 amino acids, and since each codon code for only one amino acids this means that, there are more than one code for the same amino acid. These type of codons are also called as synonymous codon . 21

22. 2. Commaless code the message is read in a continuing sequence of nucleotide triplets until a translation stop codon is reached 3. Nonoverlapping code The codon is read in mRNA in a contiguous fashion. A nucleotide that forms part of a triplet never forms part of the next triplet Each triplet is read from 5’ to 3’ direction. 5’ AUGUCUCCA 3’ 5’ -AUG-UCU-CCA-3’ : Methionine, Serine, Proline 22

23. 4 . Degeneracy Most of the codons except methionine (AUG) and trytophan (UGG) can decode the same amino acid , hence the genetic code is  degenerate.  Most of this degeneracy involves the third nucleotide of a codon. For e.g. threonine is coded by four codons ACU , ACC, ACA and ACG . 5. Unambiguity One codon codes for only one amino acid, hence, it is unambiguous and specific . 23

24. Universality of code The genetic code is largely universal for all living organisms . few exception are found in mitochondria. Where, AUA code for Methionine and UGA , one of the termination codons, code for tryptophan in yeast mitochondria . AGA and AGG code for Arginine in cytoplasm but in mitochondria they are termination codons. 7. Terminating Codon/ Non Sense Codon There are 3 codons out of 64 in genetic code which do not encode for amino acid. These are called termination codons or stop codons or nonsense codons . The stop codons are UAA , UAG and UGA . The ribosome pauses and falls off the mRNA . 8. Initiator Codon AUG is the initiator codon in majority of proteins 24

25. One tRNA can recognize more than one codon: Wobble hypothesis The first two bases of an mRNA codon always form strong Watson-Crick base pairs with the corresponding bases of the tRNA anticodon and confer most of the coding specificity. The first base of anticodon determines the number of codons recognized by the tRNA . For e.g : When the first base of the anticodon is C or A only one codon is recognized by that tRNA . When the first base is U or G, two different codons may be read. When an amino acid is specified by several different codons , the codons that differ in either of the first two bases require different tRNAs . A minimum of 32 tRNAs are required to translate all 61 codons (31 to encode the amino acids and 1 for initiation). 25

26. 26 Polypeptide synthesis - Translation

27. Steps in Translation Process Activation of Amino acid / Charging of t-RNA Initiation Elongation Termination Post translational modifications 27

28. Aminoacyl-tRNA synthetase esterify the 20 amino acids to their corresponding tRNAs at the expense of ATP energy, using Mg ++ . When attached to their specific amino acid, the tRNAs are said to be charged. Aminoacyl-tRNA synthetases accomplishes two things: 1. Activates the amino acid for polymerization 2. tRNA confers specificity by pairing with the mRNA codon Stage 1: Amino acid activation 28

29. Stage 2 : Initiation ( specific amino acid initiates protein synthesis) Protein synthesis begins at the amino-terminal end and proceeds by the stepwise addition of amino acids to the carboxyl-terminal end of the growing polypeptide There are 2 methionyl tRNAs in prokaryotic organisms : tRNA f or tRNA fmet for formyl-methionine tRNA m or tRNA met for methionine There is only one methionine aminoacyl tRNA synthetase 29

30. N- formylmethionyl tRNA fMet ( fMet-tRNAfMet ) arrives at the ribosome. Addition of N- formyl group to the amino group of methionine by the transformylase prevents fMet from entering interior positions in a polypeptide while also allowing fMet-tRNAfMet to be bound at specific ribosomal initiation P site that accepts neither Met- tRNAMet nor any other aminoacyl-tRNA . 30

31. Stage 3: peptide bonds are formed in the elongation stage The third stage of protein synthesis is elongation which requires: Initiation complex Aminoacyl-tRNAs A set of three soluble cytosolic proteins called the elongation factors( EF- Tu , EF-Ts and EF-G) and GTP   Cells use the three steps to add each amino acid residues, and the steps are repeated as many times as there are residues to be added. 31

32. Stage 4: Termination of polypeptide synthesis Elongation continues until the ribosome adds the last amino acid coded by the mRNA Termination , the fourth stage of polypeptide synthesis, is signaled by the presence of one of three termination codons in the mRNA (UAA, UAG, UGA) immediately following the final coded amino acid. 32

33. tRNA charging by aminoacyl-tRNA synthetases 33 Stage 1: Amino acid activation

34. specificity A specific N-formyl-methionine tRNA 34 Stage 2 : Initiation ( specific amino acid initiates protein synthesis)

35. ribosome distinguish start AUG and internal AUG ? Shine- Delgarno Sequence prokaryotic translation start? AUG is the Start Codon 35

36. 30S ribosomal subunit Shine- Dalgarno Sequence Recognized? The sequence of the 16 S rRNA features a short region of complementarity with the Shine/ Delgarno sequence. 36

37. The initiation of polypeptide synthesis in bacteria requires The 30S ribosomal subunit The mRNA coding for the polypeptide to be made The initiating fMet-tRNA A set of three proteins called initiation factors ( IF-1, IF-2, IF-3) GTP The 50S ribosomal subunit Mg ++ 37 formation of initiation complex in prokaryotes

38. Initiation occurs in three steps step 1 30S ribosomal subunit binds two initiation factors IF-1 and IF-3. . Factor IF-1 binds at the A site and prevents tRNA binding at this site during initiation. Factor IF-3 prevents the 30S and 50S subunits from combining prematurely. The mRNA then binds to the 30S subunit. The initiating (5’) AUG is guided to its correct position by the Shine Dalgarno Sequence in the mRNA. 38

39. Shine Dalgarno Sequence is an initiation signal of 4 to 9 purine residues. The sequence base-pairs with a complementary pyrimidine-rich sequence near the 3’ end of the 16S rRNA of the 30S ribosomal subunit. 39

40. This mRNA- rRNA interaction positions the initiating (5’) AUG sequence of the mRNA in the precise position on the 30S subunit where it is required for initiation of translation. The particular (5’) AUG where fMet-tRNA is to be bound is distinguished from other Methionine codons by its proximity to the Shine Dalgarno sequence in the mRNA. 40 Bacterial ribosomes have three sites that bind tRNAs -aminoacyl (A) site -the peptidyl (P) site and -exit (E) site the A and P sites bind to aminoacyl tRNAs whereas E site binds only to uncharged tRNAs that have completed their task on the ribosome.

41. The initiating (5’) AUG is positioned at the P site, the only site to which fMet-tRNA can bind. The fMet-tRNA is the only aminoacyl- tRNA that binds first to the P site; during the subsequent elongation stage, all other incoming aminoacyl- tRNAs bind first to the A site and only subsequently to the P and E sites. -E site is the site from which the “uncharged” tRNAs leave during elongation 41

42. 2. step 2 - initiation process, the complex consisting of the 30S ribosomal subunit, IF-3 and mRNA is joined by both GTP-bound IF-2 and the initiating fMet-tRNA . The anticodon of this tRNA now pairs correctly with the mRNA’s initiation codon. 42

43. 3. step 3 - of initiation process, this large complex combines with the 50S ribosomal subunit; simultaneously, the GTP bound to IF-2 is hydrolyzed to GDP and Pi, which are released from the complex. All these initiation factors depart from the ribosome at this point. Completion of the steps in figure produces a functional 70S ribosome called the initiation complex, containing the mRNA and the initiating fMet-tRNA . 43

44. The correct binding of the fMet-tRNA to the P site in the complete 70S initiation complex is assured by 3 points of recognition and attachment that are; 1) The codon-anticodon interaction involving the initiation AUG fixed in the P site 2) Interaction between Shine Dalgarno sequence in the mRNA and the 16S rRNA 3) Binding interaction between the ribosomal P site and fMet-tRNA The initiation complex is now ready for elongation.   44

45. 45 Formation of Initiation complex Fig: formation of initiation complex in prokaryotes

46. Stage 3: Peptide bonds formation in elongation stage The third stage of protein synthesis is elongation which requires: Initiation complex Aminoacyl- tRNAs A set of three soluble cytosolic proteins called the elongation factors( EF- Tu , EF-Ts and EF-G) and GTP Cells use the three steps to add each amino acid residues, and the steps are repeated as many times as there are residues to be added. 46

47. Steps in elongation Elongation step 1: Binding of an incoming aminoacyl- tRNA Elongation step 2: peptide bond formation Elongation step 3: Translocation 47

48. Elongation step 1: Binding of an incoming aminoacyl-tRNA appropriate incoming aminoacyL-tRNA binds to a complex of GTP-bound EF-Tu. Resulting aminoacyl - tRNA - EFTu -GTP complex binds to the A site of the 70S initiation complex. The GTP is hydrolyzed and EF- Tu -GDP complex is released from the 70 S ribosome. The EF- Tu -GTP complex is regenerated in a process involving EF-Ts and GTP . 48

49. Elongation step 2: peptide bond formation A peptide bond is now formed between the two amino acids bound by their tRNAs to the A and P sites on the ribosomes. This occurs by the transfer of the initiating N- formyl methionyl group from its tRNA to the amino group of the second aminoacyl-tRNA in the A site, forming a dipeptidyl-tRNA . 49

50. At this stage, both tRNAs bound to the ribosome shifts position in the 50S subunit to take up a hybrid binding state. The uncharged tRNA shifts so that its 3’ and 5’ ends are in the E site. Similarly, peptidyl-tRNA shift to the P site. Peptidyl transferase that catalyzes peptide bond formation is 23S rRNA ribozyme. 50

51. Elongation step 3: T ranslocation the final step of elongation cycle , the ribosome moves one codon towards the 3’ end of the mRNA. Using energy provided by hydrolysis of GTP bound to EF-G (translocase) This movement shifts the anticodon of the dipeptidyl- tRNA , from the A site to the P site, and shifts the deacylated tRNA from the P site to the E site, from where tRNA is released into the cytosol . 51

52. The third codon of mRNA now lies in the A site and the second codon in the P site. After translocation, the ribosome, with its attached dipeptidyl- tRNA and mRNA, is ready for the next elongation cycle and attachment of a third amino acid residue. 52

53. Addition of next residue occurs in the same way as addition of the second residue. The polypeptide remain attached to the tRNA of the most recent amino acid to be inserted. The existing ester linkage between the polypeptide and tRNA is broken during peptide bond formation, the linkage between the polypeptide and the information in the mRNA persists, because each newly added amino acid is still attached to its tRNA . 53

54. Stage 4: Termination Of Polypeptide Synthesis Elongation continues until the ribosome adds the last amino acid coded by the mRNA Termination , the fourth stage of polypeptide synthesis, is signaled by the presence of one of three termination codons in the mRNA (UAA, UAG, UGA) immediately following the final coded amino acid. 54

55. Once a termination codon occupies the ribosomal A site, three termination factors or release factors- the proteins RF-1, RF-2 and RF-3 contribute to: Hydrolysis of the terminal peptidyl tRNA bond Release of the free polypeptide and the last tRNA , now uncharged, from the P site and Dissociation of 70 S ribosome into its 30S and 50S subunits, ready to start a new cycle of polypeptide synthesis. 55 RF-1 recognizes the termination codons UAG and UAA. Rf-2 recognizes UGA and UAA. Either RF-1 or RF-2 binds at a termination codon and induces peptidyl transferase to transfer the growing polypeptide to a water molecule rather than to another amino acid. The specific function of RF-3 has not been firmly established.

56. Hydrolysis of GTP by EF-G leads to dissociation of the 50S subunit from the 30S-tRNA –mRNA complex. The complex of IF-3 and 30S subunit is then ready to initiate another round of protein synthesis. EF -G and ribosome recycling factor( RRF ) are replaced by IF-3 which promotes the dissociation of tRNA . The mRNA is then released. 56

57. STAGE 5: Post Translational Modifications Folding And Processing It is a final stage of protein synthesis where nascent polypeptide chain is folded and processed into its biologically active form. Some newly made proteins donot attain their final biologically active conformation until they have been altered by one or more processing reactions called post translational modifications . 57

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60. Amino-terminal and carboxyl-terminal modification: The first residue inserted in all polypeptides is N – formylmethionine (in bacteria) or methionine (in eukaryotes). The formyl -group, the amino terminal Met residue and additional amino terminal residues may be removed enzymatically to form final functional protein. 2. Formation of disulfide cross-linkage: After folding into their native conformations, some proteins form intrachain or interchain disulfide bridges between Cys residues. This disulfide cross-linkage help to protect the protein conformation from denaturation in the extracellular environment. 60

61. 3. Loss of signal sequences: 15-30 residues at the amino terminal end of some proteins called signal sequences which play a role in directing the protein to its ultimate destination in the cell are removed by specific peptidases. 4 . Modification of individual amino acids: The hydroxyl groups of certain amino acids (Ser, Thr , and Tyr) are enzymatically phosphorylated by ATP adding more negative charge to polypeptide. The functional significance of this modification varies from one protein to the next. For e.g : milk protein casein has many phosphoserine groups that bind Ca 2+ 61

62. 5. Attachment of carbohydrate side chains/ Glycosylation : The carbohydrate side chains of glycoproteins are attached covalently during or after polypeptide synthesis. The complex carbohydrate moiety is attached to the amino acids, serine and threonine (O- linked) or to asparagine (N-linked), leading to the synthesis of gylcoproteins . Lubricating proteoglycans that coat mucous membrane, contains oligosaccharide side chains. 62

63. 6. Addition of isoprenyl groups The isoprenyl groups are derived from pyrophosphorylated intermediates of the cholesterol biosynthestic pathway such as farnesyl pyrophosphate. The isoprenyl group helps to anchor the protein in a membrane. The carcinogenic activity of the ras oncogene is lost when isoprenylation of the Ras protein is blocked. This has stimulated interest in identifying inhibitors of this posttranslational modification pathway for use in cancer chemotherapy . 63

64. 7. Addition of prosthetic groups : Many proteins require covalently bound prosthetic groups. Two examples are the biotin molecule of acetyl-CoA carboxylase and the heme - group of hemoglobin or cytochrome c . 8. Proteolytic procesing : Many proteins are initially synthesized as large, inactive precursor of polypeptides that are proteolytically trimmed to form their smaller, active forms. Examples include formation of insulin from preproinsulin , some viral proteins, and proteases such as chymotrypsinogen and trypsinogen . 64

65. Significance Of Translation Process Translation is the ultimate step of gene expression by which the information of DNA carried by mRNA is decoded in the forms of protein. It is also a detection tool in genetic recombinant technology by which DNA of desired character is found to be present in cDNA or not by expression selection. It is the tool by which proteins are synthesized from the amino acids. 65

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67. 2077-03-19 Utpal 67

68. Explain the process of translation (Prokaryotic model) in detail. (using diagrams is preferable) 68