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Protein Metabolism

Protein Metabolism. Protein Synthesis. Protein Synthesis: an overview. PS proceeds from N -terminus to C-terminus (amino to carboxyl) Ribosomes:read mRNA in 5’ to 3’ direction Polyribosomes (polysomes) Max. density ~ 1 ribosome/80 nucleotides. Protein Synthesis: an overview.

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Protein Metabolism

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  1. Protein Metabolism Protein Synthesis

  2. Protein Synthesis: an overview • PS proceeds from N -terminus to C-terminus (amino to carboxyl) • Ribosomes:read mRNA in 5’ to 3’ direction • Polyribosomes (polysomes) • Max. density ~ 1 ribosome/80 nucleotides

  3. Protein Synthesis: an overview • Chain elongation: links the growing chain to incoming tRNA’s AA residue • AA residues: added to C-terminus • Ribosome (prokaryote) has 3 tRNA binding sites • The P site: peptidyl site • Binds peptidyl –tRNA • Peptidyl-tRNA holds polypeptide • After peptide bond formation, binds deacylated tRNA • The A site: amino-acyl site • Binds incoming aminoacyl-tRNA

  4. Protein Synthesis: an overview • Ribosome (prokaryote) has 3 tRNA binding sites • After peptide bond formation • Deacylated tRNA released from P site • Replaced in P site by newly formed peptidyl-tRNA • A site is vacated • The E site: exit site • Recent finding • Largely confined to 50S subunit • Deacylated tRNA dissociate from the ribosome

  5. Protein Synthesis: an overview • Five (5) major stages of protein synthesis • 1.Activation of Amino Acids • 20 AA • 20 aminoacyl-tRNA synthetases • 20 or more tRNAs (min 32) • ATP • Mg2+

  6. Protein Synthesis: an overview • Five (5) major stages of protein synthesis • 2.Initiation • mRNA • N-Formylmethionyl-tRNA • Initiation codon in mRNA (AUG) • Ribosome: 30S and 50S subunit • Initiation factors: IF-1. IF-2, IF-3 • GTP • Mg2+

  7. Protein Synthesis: an overview • Five (5) major stages of protein synthesis • 3.Elongation • Initiation complex (functional 70S ribosome) • aminoacyl-tRNAs specified by codons • Peptidyl transferases • Elongation factors: EF-Tu. EF-Ts, EF-G • GTP • Mg2+

  8. Protein Synthesis: an overview • Five (5) major stages of protein synthesis • 4. Termination and Release • Termination codon in mRNA • Polypeptide releasing factor: RF1, RF2, RF3 • ATP • 5. Folding and Processing • Enzymes (lots!) • Cofactors (lots!)

  9. P S: stage one; activation of AA • Takes place in the cytosol • Each AA is attached to specific tRNA • ATP to AMP + Ppi • Catalyzed by MG 2+ -depending aminoacyl-tRNA synthetases • Aminoacylated tRNA is said to be charged

  10. P S: stage two; Initiation • Initiation codon recognized by tRNAfmet • N-formylmethionine residue (fmet) • Special tRNA • Special aminoacyl-tRNA synthetase • First: bind tRNAfmet with Met • Then: N –formulates the Met residue • Note: proteins are post-translationally modified • Deformylation of fmet residue • Sometimes: removal of N-terminal Met

  11. P S: stage two; Initiation • Initiation codon recognized by tRNAfmet • In eukaryotes: • All polypeptides synthesized by cytoplasmic ribosomes • Begin with Met residues (not fmet) • Have special initiating tRNA • Mitochondrial/chloroplast products are like polypeptides

  12. P S: stage two; Initiation • The Shine-Dalgarno Sequence: • John Shine and Lynn Dalgarno in 1974 • An initiating signal in mRNA • 8-13 bp to the 5’ side of initiation codon • 4-9 purine residues • Base pair with (antiparallel): • Complementary pyrimidine-rich sequence • Nearnear 3’ end of 16SrRNA on 30S subunit • mRNA-rRNA interactions • Sets mRNA in the correct position • Initiation of transcription

  13. P S: stage two; Initiation • Three Stages: assemble Initiation Complex • Requires Initiation factors • Not permanently associated with ribosome • 3 (in E. coli ): IF-1, IF-2, IF-3 • Stage one (1): • 30S subunit binds IF-3, IF-1 • Prevents premature joining of LG and SM subunits • mRNA binds to 30S • Initiation codon (AUG) to P-site on 30S subunit • Guided by Shine-Delgarno sequence

  14. P S: stage two; Initiation • Three Stages: assemble Initiation Complex • Stage two (2): • 30S subunit with IF-3 with mRNA binds w/IF-2 • IF-2 is already bound to: • GTP • fMet-tRNA fmet • Anticodon and codon pair

  15. P S: stage two; Initiation • Three Stages: assemble Initiation Complex • Stage three (3): • Large complex formed in stage 2 combines with 50S subunit • GTP to GDP and Pi • IF-3, IF-1, IF-2 are released • IF-3 released before 50S attaches • irreversible

  16. P S: stage two; Initiation • Three Stages: assemble Initiation Complex • At end of Initiation: • fMet-tRNA fmet with mRNA with Ribosome complex is formed • fMet-tRNA fmet in P site • A site ready • Eukaryotic Initiation is similar • More initiation factors • No Shine-Dalgarno • Cap is located; the 1st AUG downstream

  17. P S: stage three; Elongation • 3 stage cycle that is repeated • Adds AA to C-terminus • Up to 40 residues/sec • Elongation factors (EF)

  18. P S: stage three; Elongation • Aminoacyl-tRNA binding • GTP with EF-Tu with aminoacyl-tRNA binds to ribosome • aminoacyl-tRNA: bound in codon-anticodon interaction at A site • GTP to GDP + Pi • EF-Tu to GDP + Pi are released • Regenerate GTP • EF-Tu with GDP + EF-T3 to EF-Tu with EF-Ts + GDP • EF-Tu with EF-Ts + GTP to EF-Tu with GTP + EF-Ts

  19. P S: stage three; Elongation • Transpeptidation: • Peptide bond formation • Transfer of N-formylmethionyl group • From tRNA in P site • To amino group of 2nd AA in A site • Forms a dipeptidyl-tRNA in A site • tRNA fmet in P site • Peptidyl transferases • Catalyzes bond formation on LG subunit • Catalyzed by 23S rRNA (Harry Noller, 1992)

  20. P S: stage three; Elongation • Translocation: • Ribosomes moves toward 3’ end by one codon • Dipeptidyl-tRNA moves to Psite • Deacylated- tRNA fmet released • New codon (3rd) into A site • Shift requires • EF-G (translocase) • GTP • Believed to be accompanied by 3-D changes in ribosome

  21. P S: stage three; Elongation • Repeat elongation cycle • Need 2 GTP for each added AA residue • Protein chain always remains attached to a tRNA

  22. P S: stage four; TERMINATION • Signaled by termination codon • When termination codon is in the A site • 3 releasing factors • RF1, RF2, RF3 • Hydrolysis of terminal peptidyl-tRNA bond • Release of protein and last tRNA • Dissociation of ribosome

  23. P S: stage four; TERMINATION • When termination codon is in the A site • 3 releasing factors • RF1: reacts to UAG, UAA • RF2: reacts to UGA, UAA • RF3: ? • RF bind at termination codon • Peptidyl transferase gives chain to H2O • Eukaryotes • One RF: eRF • Recognizes all 3 termination codons

  24. P S: stage five; post-translational modification • To become mature, polypeptides must fold to native conformations • Disulfide bonds must form • Multisubunit proteins: subunits must combine • Must be modified by enzymes • Proteolytic cleavage • Most common P-T modification • Eg: all proteins have fMet residue removed • Eg: Conversion of trypsinogen to trypsin

  25. P S: stage five; post-translational modification • Covalent modifications • E.g.: • Methylations • Hydroxylations • Etc • Changes in functional groups and radical groups

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