Protein Translation- cell molecular biology.pptx
- 1. Translation Initiation, elongation and termination phases of translation, actual process involving different enzymes/protein factors; posttranslational processing and translational inhibitors
- 2. Ribosome • The smaller SU contains 16S rRNA and 21 different proteins • The larger one has 5S and 23S rRNA with 31 proteins • Proteins are designated by the prefixes S or L followed by a number that roughly increases from the largest to the smallest • Proteins range from 45 to 557 residues and a single copy of each occur/ribosome with exception of L12, 4 copies of which occurs/ribosome • Possess little sequence similarity among them and are rich in Lys and Arg and Ar- amino acid residues • E. coli cells having around 20,000 ribosomes and account for ~80% of its RNA and 10% of its protein contents
- 3. • Larger and smaller SU contacts each other via 12 sites involving RNA-RNA, Protein-protein and RNA-Protein interaction. Mg+ ions very often mediate many of these interactions • Has three tRNA binding sites: – A-site, binds the incoming aminoacylated tRNA (smaller SU) – P-site, binds the peptidyl tRNA (smaller SU), and – E-site, binds the uncharged tRNA (Larger SU)
- 6. • Initiator tRNA is fMet-tRNAfMet • Formyl donor is N10 formyl FH4 • Formylation catalyzed by a specific transformylase that can’t recognize Met-tRNAfMet • The 16S rRNA has a pyrimidine rich sequence at its 3’ end known as Shine-Dalgarno sequence, partially complementary to and base pairs with a purine rich tract centered at ~10 NT upstream from the start codon AUG of the mRNA
- 8. • ORF? • RBS? • Shine-Dalgarno sequence?
- 10. • IF3 binds to 30S SU • mRNA and IF2 in a ternary complex with GTP and fMet-tRNAfMet along with IF1 subsequently bind to the 30S SU • IF1 blocks the A site to prevent binding any incoming aminoacyl tRNA there
- 11. • 50S SU now joins the 30S initiation complex releasing IF1, IF3 and IF2 promoting hydrolysis of its bound GTP into GDP+Pi forming 70S initiation complex
- 12. • Decoding: Ribosome recoignizes and binds the aminoacyl tRNA at the ‘A’ site and form complementary base pairing between anticodon of tRNA and the codon on the mRNA
- 13. • Transpeptidation: Peptidyl group at the P-site is transferred to the aminoacyl group at the A-site through formation of a peptide bond
- 15. • Translocation: Ribosome moves by one codon triplet downstream w.r.t. the mRNA with its bound A-site and P-site tRNAs
- 17. RF1 recognizes UAA and UAG, whereas RF2 UAA and UGA, Release factors bring about the hydrolysis of the polypeptide- tRNA link yielding an uncharged tRNA in the P site, a free polypeptide chain that dissociates from ribosome RF3 binds with ribosome in combination with GTP and its subsequent hydrolysis releases RF1 and RF2
- 18. RF3-GDP complex is released and then ribosome recycling factor (RRF) binds in the A-site followed by EFG- GTP. EFG hydrolyzes its bound GTP causing RRF to move to the P-site and the tRNAs occupying the P- and E- sites to be raleased Finally, the RRF and EFG-GDP and mRNA dissociate yielding an inactive 70S ribosome ready for reinitiation
- 19. Posttranslational processing The nascent polypeptide molecule undergo one or more processing steps to progressively attain its native 3D conformation needed for biological activity
- 20. N- & C-terminal modifications: a) Removal of the N-formyl group b) Removal of the fMet or additional residues c) C-terminal residues may also be removed/modified Loss of signal sequences: ~15-30 N-terminal residues being the signal required for directing protein to its ultimate destination in the cell may be removed after the purpose is served Attachment of carbohydrate side chains: Carbohydrate side chains added to Asn (N-linked) or Ser or Thr (O-linked) residues of glycoproteins; many function as extracellular lubricating proteoglycans that coat mucous membranes contain oligosaccharide side chains
- 21. Modification of the individual amino acid residues: a) OH of Ser, Thr & Tyr of some proteins are enzymatically phosphorylated by ATP; e.g. phosphoserine residues of casein b) Additional COOH groups may be added to Glu residues of some proteins; e.g., N- terminal ɣ-COOH Glu residues of prothrombin for binding Ca2+ needed for blood clotting c) Mono- & dimethyllysine residues occur in some muscle proteins and in cytochrome c; trimethyllysine residues at specific position in calmodulin d) COOH groups of some Glu residues undergo methylation sheilding the negative charge
- 22. Addition of isoprenyl groups Attachment of isoprenyl groups through a thioether bond with Cys residue of the protein. The isoprenyl groups are derived from pyrophosphorylated intermediates (e.g., farnesyl pyrophosphate) of cholesterol biosynthesis pathway; e.g., Ras proteins, products of the ras oncogenes and proto-oncogenes, G proteins, lamins (proteins found in nuclear matrix) Addition of prosthetic groups Addition of covalently bound prosthetic groups; e.g., heme group in hemoglobin Proteolytic processing Many nascent proteins are proteolytically trimmed in specific positions yielding their smaller active forms from the inactive precursors; e.g., proinsulin to insulin Forming disulfide cross links After folding some proteins form disulfide cross links between Cys residues in specific positions
- 23. Translational inhibitors Puromycin, an inhibitory antibiotic produced by Streptomyces alboniger, resembles 3’ end of an aminoacyl tRNA, binds to A- site of prokaryotic ribosome, form peptide bond, but the peptidyl puromycin inhibits translocation and dissociation from ribosome
- 25. Tetracyclines inhibit bacterial translation; blocks the A-site preventing access to aminoacyl tRNAs Chloramphenicol inhibits bacterial, mitochondrial and chloroplast translation; blocks peptidyl transfer; doesn’t affect cytosolic translation in eukaryotes Cycloheximide blocks transpeptidation of 80S (eukaryotic) ribosomes but not 70S bacterial (and mitochondrial and chloroplast) ribosomes
- 26. Streptomycin, a basic trisaccharide, causes misreading of genetic code (in bacteria) at low concentrations and inhibits initiation at higher concentrations Diptheria toxin, catalyzes an ADP-ribosylation reaction inactivating eukaryotic elongation factor eEF2 Ricin, a toxic protein of castor bean depurinates a specific adenosine residue in 23S rRNA and inactivates the 60S subunit of eukaryotic ribosomes