translation- ppt class 12 DAV public School Chandrasekharpur

ppt on transcription

1. TRANSLATION By- sunandan

2. Translation is the process of protein synthesis in which the genetic information encoded in mRNA is translated into a sequence of amino acids in a polypeptide chain   Proteins are made up from a set of 20 amino acids called standard amino acids. Each of the 20 amino acids is specified by specific codons Polypeptide synthesis proceeds from N-terminus to C-terminus and ribosome read in 5’-3’ direction LOCATION The translation takes place on ribosome in cytoplasm of the cell. It is simultaneous in prokaryotic transcription, while required processing mRNA in eukaryotes

3. COMPONENTS REQUIRED FOR TRANSLATION RIBOSOMES In prokaryotes ribosomes are distributed through out the cells In eukaryotes ribosomes are located in the cytoplasm frequently on the extensive intra cellular membrane network of the endoplasmic reticlum The ribosomes of eukaryotes are larger, usually about 80S how ever size varies from species to species The ribosome present in the mitochondria and chloroplast of eukaryotic cells are smaller usually about 60S Ribosomes are approximately half protein and half RNA

4. A ribosome is composed of two halves, a large and a small subunit. During translation, ribosomal subunits assemble together like a sandwich on the strand of mRNA Each subunit is composed of RNA molecules and proteins The small subunit binds to the mRNA The large subunit has binding sites for tRNAs and also catalyzes peptide bonds between amino acids TRANSFER RNAs The translation of a coded mRNA message into a sequence of amino acids in a poly peptide requires tRNA molecule Atleast one specific type of tRNA is required per amino acid. Also a single tRNA may recognize two or three codons all specifying one single amino acid

5. Anti codon arm of tRNA contain a triplet nucleotide sequence, which is complementary to and base pairs with the codon sequence in mRNA during translation Because of tRNAs ability to both carry a specific amino acid and to recognize the codon for that amino acid, tRNAs are also known as adaptor molecule tRNA binding sites on ribosomes A or amino acyl site  binds the incoming aminoacyl tRNA , the tRNA carrying the next amino acid to be added to the growing polypeptide chain P or peptidyl site binds the tRNA to which the growing polypeptide is attached E or exit site binds the departing uncharged tRNA

6. Messenger RNA The specific mRNA required as a template for the synth e sis of the desired polypeptide chain must be present . Amino acids All amino acids that appear in the finished protein must be present at the time of protein synthesis. ATP and GTP ATP and GTP are required as the source of energy. Protein factors Initiation, elongation, termination/release factors are required for peptide synthesis.

7. STEPS IN TRANSLATION Activation of aminoacid /charging of tRNA Polypeptide chain Initiation Chain elongation Chain termination

8. Activation of Amino acid The attachment of amino acids to tRNAs is the function of the group of enzymes called aminoacyl - tRNA synthetases (also known as aminoacyl tRNA ligase ) aminoacyl - tRNA synthetases activate the amino acids by covalently linking them to tRNAs When a tRNA is chared with the amino acid corresponding to its anti codon it is called aminoacyl - tRNA Amino acylation reaction is performed by two step process Amino acids are activated by ATP forming an intermediate amino acyl adenylate Amino acid is transferred to the 3’ end of the tRNA Amino acid attachment site The amino acids are attached to the tRNAs by high energy bond ( ester bond ) between the carboxyl group of the aminoacids and 3’ hydroxyl termini of the tRNA

9. There are 20 aminoacyl-tRNA synthetases for 20 amino acids. These fall into two distinct groups class I &class II Class I  these attach the aminoacid to the free 2’or 3’ hydroxyl groups of the adenosine at the 3’ terminus of tRNA molecules by ATP requiring reaction.these are generally monomeric . Class II  these attach to the 3’OH group .these are generally dimeric or multimeric

10. Translation in Prokaryotes

11. Polypeptide chain initiation Initiation involves binding of small ribosomal subunit to mRNA and then is joined by the 50S subunit .it involves the reactions that precede the formation of the peptide bond between the first two amino acids of the protein Major factors that are needed for initiation Initiating amino acid and initiating codon The synthesis of all proteins starts with the same amino acid Methionine in E.coli and in other Eubacteria the first amino acid in any newly synthesized polypeptide is N- formyl methionine In eubacteria such as E.coli , AUG and GUG and on rare occasions UUG , serve as initiation codons these are present in the initiating positions and these are recognized by N- formyl met tRNA so first amino acid appeared is formyl methionine The meaning of AUG and GUG codons depends on their context. AUG  ( Initiating position  formyl methionine , coding region  methionine )

12. GUG  ( Initiating position formyl methionine , coding region  Valine ) Initiating tRNA In prokaryotes the initiator tRNA carries a formylated methionine residue that initiates the protein synthesis.it recognize the initiating codons AUG,GUG,UUG Conserved feautures of initiator tRNA distinguish them from elongator tRNA Absence of base pairing between the bases 1 and 72 in acceptor arm Presence of three consecutive GC base pairs in anticodon arm(these base pairs are required to allow the initiator tRNA to be inserted directly in to the P-site

13. Initiation factors IF-1  binds over the A site of small ribosomal subunit and is thought to prevent the initiator tRNA from binding to the A-site IF-2  directs the initiator tRNA to its correct position in the initiation complex.it has ribosome dependent GTPase activity. The GTP is hydrolyzed when the 50S subunit joins to generate a complete ribosome IF-3  binds to E site of 30S subunits and prevent premature reassociation of the large and small subunits of the ribosome and also controls the ability of 30S subunits to bind to mRNA and also selects the initiator tRNA for use in initiation Shine Dalgarno sequence The 30S subunit of the ribosome contains 16SrRNA The 3’ end of the 16SrRNA contains a consensus sequence called shine dalgarno sequence and it is complementary to the 5’end sequence present in the mRNA When they are in contact with the each other correct positioning of mRNA is done

14. Steps involved in initiation Binding of IF-3 to the E site this prevents the premature binding of 50S subunit to the 30S subunit Binding of IF-1 to the A site this prevents premature reassociation amino acyl tRNA to the A site mRNA gets associated with the 30S subunit of the ribosome. Correct positioning is said that when shine dalgarno sequence of 16S rRNA binds to the 5’ region of the mRNA GTP bound IF-2 recruits the f met tRNA to the P site of the 30S subunit of the ribosome GTP hydrolysis occurs and removal of all factors have been done Joining of the 50S subunit and form 70S initiation complex

15. Chain Elongation Elongation is a cyclic process on the ribosome in which one aminoacid at a time is added to the nascent peptide chain Elongation rate in E.coli is roughly 15 amino acids per second Major factors that are needed for initiation: EF- Tu : Directs the next tRNA to its correct position in the ribosome. EF-Ts : Regenerates EF- Tu after the hydrolysis of attached GTP molecule EF-G: Mediates Translocation Elongation takes place in 3 stages Decoding: In this case the ribosome selects and binds an amino acyl tRNA whose anti codon is complementary to the mRNA codon in the A site Transpeptidation : During tranpeptidation the peptidyl group on the P site of tRNA is transferred to the amino acyl group in the A site through the formation of peptide bond Translocation: In translocation, A and P site tRNAs are respectively transferred to the P site and E site accompanied by their bound mRNA

16. Steps involved in Elongation: EF- Tu with GTP mediates the entry of amino- acyl - tRNA into the A site and forms EF- Tu GTP complex and this binds with the tRNA . next hydrolysis of GTP of the complex to GDP helps drive the binding of amino- acyl tRNA to the A site at which EF- Tu is released and it becomes inactivated and it is activated by EF-Ts The carboxyl end of the polypeptide chain is uncoupled from the tRNA molecule in the P site and joined by a peptide bond to the amino acid linked to the tRNA molecule in the A site. This reaction of protein synthesis is catalyzed by a peptidyl transferase . The primary activity of peptidile transfer however is a ribozyme activity encoded in the 23S rRNA of larger sub unit The t RNA present in the A site moves to the P site and the tRNA present in the P site moves to E site. This is performed by EF-G (Also called Translocase ) with GTP by hydrolysing into GDP

17. Third step in elongation

18. Proofreading Two fundamental different proofreading mechanisms are used in the cell. Both are active process I) Chemical proofreading This is carried out by amino acyl tRNA synthetases which recognizes an incorrect amino acid attached to its tRNA molecule and remove it by hydrolysis When incorrect amino acid binds to synthetases and form non cognate tRNA adenylate then the wrong amino acid is added to the tRNA , is then recognized as incorrect as incorrect by its structure in the tRNA binding site, and so is hydrolyzed and released. II) kinetic proofreading An incorrect tRNA molecule forms a smaller number of codon-anticodon hydrogen bonds than a correct one, it there fore binds more weakly to the ribosomes and is more likely to dissociate during the period where elongation EF-G hydrolyses its GTP The elongation factor thereby introduces a short delay between codon-anticodon basepairing and polypeptide chain elongation, which provides an opportunity for the bound tRNA molecule to exit from the ribosome Thus the delay introduced by the elongation factor causes most incorrectly bound tRNA molecules to leave the ribosome with out being used for protein synthesis. ……

19. Chain Elongation

20. Chain termination Protein syhthesis ends when one of the three termination (stop) codons (UAA,UAG,UGA) is reached. Major factors that are needed for termination To terminate the polypeptide chain we need release factors(RF) and they are classsified into class I(RF1&RF2) and class II(RF3) RF-1  recognizes the termination codons 5’-UAA-3’ and 5’-UAG-3’ RF-2  recognizes 5’-UAA-3’ and 5’-UGA-3’ RF-3  stimulates dissociation of RF-1 and RF-2 from the ribosome after termination Steps involved in termination The A site is now entered not by a tRNA , but by a protein release factor RF-1 and RF-2 recognizes the stop codons and activate the ribosome to hydrolyze the bond between tRNA and polypeptide release through a GGQ( Gly-Gly-Gln ) motif which acts as peptide anticodon RF-3 stimulates release of RF-1 or RF-2 from the ribosome after termination in a reaction requiring energy from the hydrolysis of GTP

21. Chain termination Now the RRF(ribosomal release factor )along with GTP and EFG binds to the A site and acts as translocation and release the uncharged tRNA in the E site Once E site becomes free automatically IF3 binds to the E site and results in dissociation of the larger subunit and all the products are released.

22. EUKARYOTIC TRANSLATION

23. Polypeptide chain initiation Eukaryotic initiation takes place in two different ways Cap dependent initiation Cap independent initiation EXAMPLE : Cap dependent initiation  majority of eukaryotes go through cap dependent initiation EXAMPLE : Cap independent initiation  this generally go through IRES(internal ribosome entry site) ,generally viruses in eukaryotes go through this mechanism and also in cellular stress, when overall translation is reduced. Examples include factors responding to apoptosis and stress-induced responses. Cap dependent initiation Cap independent initiation Circulization of mRNA No circulization of mRNA 5’ cap of mRNA is presnt 5’ cap of mRNA is absent Poly A tail is present Poly A tail is absent Scanning of mRNA takes place No scanning of mRNA takes place

24. Cap dependent initiation Initiation in eukaryotes involves different steps The main difference between translation in eubacteria and in eukaryotes occur during the initiation phase. Eukaryotic cells require more initiation factors than eubacteria (12 factors required for initiation)

25. 43S pre initiation complex In the first step the EIF3 binds to the E site of the 40S subunit so that it can prevent the premature reasocciation of 60S subunit EIF1 binds to the A site of the 40S subunit so that P site is free and the initiator tRNA can directly enter in to it EIF2 along with GTP binds to the initiator tRNA and forms ternary complex and bring the tRNA to the P site of the 40S ribosome(but the tRNA is not properly placed in the P site due to the absence of mRNA 43S pre initiation complex

26. 48S pre initiation complex(attachment of mRNA) In eukaryotes mRNA has 5’ cap and 3’ poly A tail and also some secondary structures at 5’ end the mRNA also have contiguous nucleotide sequence of 5’-GCC(A or G)CCAUGG3’ this sequence is required for optimal initiation translation in eukaryotes which is called kozak’s rule/ kozak’s sequence For the attachment of mRNA to the 43S complex it requires EIF4 complex.(it is a heterotrimeric complex containing EIF4A,EIF4G,EIF4E ) EIF4E (acts as cap binding protein ) binds to the 7 met guanosine which is a cap region at 5’ end and next EIF4A (acts as helicase ) binds to the secondary structure at 5’ end and helps in removing the secondary structures EIF4G binds to the EIF4E and in turn PABP (poly A binding protein ) binds to the EIF4G. This leads to the circulization of poly A tail takes place Now we have both mRNA bind initiation factors and 43S initiation complex they both come and contact with each other and now it start to scan for the kozak sequence in the mRNA Scanning takes place with the help of Dhx29 and Ddx3 proteins on the mRNA

27. Once it finds the start codon proper placing of the tRNA takes place(forms complete 48S initiation complex) by hydrolyzing the GTP bound to EIF2 to EIF-GDP (this is again utilized in GTP form and this conversion takes place with the help of EIF2B)

28. 48S pre initiation complex

29. 80S pre initiation complex(assembly of larger subunit) Larger subunit 60S along with EIF5 and GTP comes and binds that removes all the factors present in the complex later GTP that is present withEIF5 also gets hydrolyzed and forms complete 80S complex 80S initiation complex

30. Chain Elongation Elongation is a cyclic process on the ribosome in which one aminoacid at a time is added to the nascent peptide chain The eukaryotic elongation cycle closely resembles that of prokaryotes factors that act similar as in prokaryotic elongation factors are EF- Tu eEF1A EF-Ts eEF1b EF-G  eEF-2 Elongation takes place in 3 stages Decoding: In this case the ribosome selects and binds an amino acyl tRNA whose anti codon is complementary to the mRNA codon in the A site Transpeptidation : During tranpeptidation the peptidyl group on the P site of tRNA is transferred to the amino acyl group in the A site through the formation of peptide bond Translocation: In translocation, A and P site tRNAs are respectively transferred to the P site and E site accompanied by their bound mRNA In Prokaryotes In Eukaryotes

31. Steps involved in chain elongation eEF1A along with GTP brings the incoming tRNA to the A site and once it brings there GTP gets hydrolyzed to GDP and again it should convert in to GTP. This is done by the complex of (EFIB,EFIG,EF1D) Next step is to add peptide bond it is done with the help of the enzyme peptidyl transferase which is performed by the rRNA of the larger ribosome.

32. In the next it is translocated three nucleotides in 5’ to 3’ direction with the help of Eef-2

33. Chain Elongation

34. Co-translational translocation Most proteins that are secretory , membrane-bound, or reside in the  endoplasmic reticulum  (ER),  golgi  or  endosomes  use the co-translational translocation pathway. This process begins with the N-terminal  signal peptide  of the protein being recognized by a  signal recognition particle  (SRP)  while the protein is still being synthesized on the ribosome . The synthesis pauses while the ribosome-protein complex is transferred to an  SRP receptor  on the ER in  eukaryotes . There, the nascent protein is inserted into the translocon , a membrane-bound protein conducting channel composed of the  Sec61 translocation complex  in eukaryotes. In secretory proteins and type I  transmembrane proteins , the signal sequence is immediately cleaved from the nascent polypeptide once it has been translocated into the membrane of the ER (eukaryotes) by  signal peptidase . The signal sequence of type II membrane proteins and some polytopic membrane proteins are not cleaved off and therefore are referred to as signal anchor sequences

35. Within the ER, the protein is first covered by a  chaperone protein  to protect it from the high concentration of other proteins in the ER, giving it time to  fold  correctly. Once folded, the protein is modified as needed (for example, by  glycosylation ), then transported to the Golgi for further processing and goes to its target organelles or is retained in the ER by various  ER retention  mechanisms

36. Post-translational translocation Even though most secretory proteins are co- translationally translocated , some are translated in the  cytosol  and later transported to the ER/plasma membrane by a post-translational system. This pathway is facilitated by  Sec62  and  Sec63 , two membrane-bound proteins. The Sec63 complex is embedded in the ER membrane. The Sec63 complex causes hydrolysis of ATP, which allows chaperone proteins to bind to an exposed peptide chain and slide the polypeptide into the ER lumen. Once in the lumen the polypeptide chain can be folded properly. This occurs in only unfolded proteins that are in the cytosol . In addition, proteins targeted to other destinations, such as  mitochondria ,  chloroplasts , or  peroxisomes , use specialized post-translational pathways. Also, proteins targeted for the nucleus are translocated post-translation. They pass through the  nuclear envelope  via  nuclear pores .

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38. Chain termination Two types specific protein release factors have been discovered eRF1(eukaryotic release factor 1)  it acts by binding to the ribosomal A site and recognizing stop codons directly eRF3  it is GTP binding protein .The eRF3-GTP acts along with eRF1 to promote clevage of the peptidyl tRNA thus releasing the completed protein chain eRF3 GTPase monitors the correct recognition of a stop codon by eRF1 Hydrolysis of eRF3-GTP to eRF3-GDP assumes correct termination of translation.

39. Chain Termination

40. Inhibitors Function Chloramphenicol Inhibits peptidyl transferase on larger subunit in prokaryote Cyclohexamide Inhibits peptidyl transferase on larger subunit in eukaryote Erythromycin Inhibits translocation in prokaryote Paromomycin Increase ribosomal error rate Puromycin It is a Amino acyl tRNA analogue (premature chain termination in both prokaryotes and eukaryotes) Streptomycin mRNA misreading and inhibits chain initiation in prokaryotes Tetracyclin Inhibits binding of amino acyl tRNA to smaller subunit in prokaryote Inhibitors of Protein Synthesis

41. Inhibitor Function Diptheria Inactivates eEF2 by ADP ribosylation Ricin / Abrin / Sarcin - (Fungal protein) Ricin and Abrin are poisinous plant glycosidases inactivate eukaryotic large subunit by depurinating the 28SrRNA located in sarcin-ricin loop that is useful for ribosomal factors binding centre Sarcin cleaves phosphodiesterase bond in sarcin-ricin loop Fusidic acid Inhibits protein synthesis by targeting ribosome bound EG-G in both translocation and ribosome recycling factor or release factor in prokaryotes