Protein Biosynthesis II mRNA Binding Shine-Dalgarno Sequence Initiation Complex f-Met-tRNA f Met Initiation factors Elongation Elongation factors Termination Termination factors mRNA Binding Shine-Dalgarno Sequence Initiation Complex f-Met-tRNA f Met Initiation factors Elongation Elongation factors Termination Termination factors

Dintzis Experiment COO - 5’ Trypsin H 2 N- COO - C-terminal is richer in radioactive 3 H-leucine C-terminal is richer in radioactive 3 H-leucine 3 H-Leucine H 2 N- 3’ Released Protein 4 min 8 min 60 min 3H3H C-terminalN Product analysis

Shine-Dalgarno Purine-rich mRNA Pyrimidine-rich rRNA complementary

fMet-tRNA f Met CH 3 -S-CH 2 CH 2 C-COO-tRNA H N-H C=O H N-Terminal Block Blocking the N-terminal amino acid assures that the peptide chain will grow towards the C-terminal Blocking the N-terminal amino acid assures that the peptide chain will grow towards the C-terminal

Vocabulary of Terms Aminoacyl-tRNA: tRNA with an amino acid attached Peptidyl-tRNA: tRNA with peptide attached Nascent chain: peptide chain in act of being assembled P-site: Site on ribosome where peptidyl-tRNA sits A-site: Site on ribosome where incoming aminoacyl-tRNA binds Peptidyl transferase: Enzyme that forms peptide bond

Protein Biosynthesis takes place on Ribosomes Picture the ribosomes as an assembly site and a ratchet machine GTP, not ATP, is the fuel that drives the machine tRNAS are brought to the assemble site called the “A” site A peptide bond is formed by transferring the peptide from the P site to the N-terminal of the new amino acid (aminoacyl-tRNA) OVERALL MECHANISM

The peptide now has the new amino acid in the C-terminal…still attached to the tRNA The peptidyl tRNA is in the A site The mRNA with the peptidyl-tRNA attached shifts one frame bringing a new codon into the A site Simultaneously, the peptidyl tRNA is pushed into the P site and the uncharged tRNA is shoved into the E site and expelled (OVERALL CONT.)

1. The machine is disassembled into its component parts to allow the mRNA to bind 2. The starting tRNA is set into position 3. The larger 50S subunit is put back in place INITIATION

Initiation Complex

Elongation 2. An aminoacyl-tRNA reacts with elongation factor EF-Tu in preparation for binding to the A site. 1. Peptidyl-tRNA sits in the P site 3. A-site binding occurs as GTP is hydrolyzed. EF-Ts removes spent GDP. 4. A peptide bond is formed with the transfer of the peptide from the P site to the A site 5. Elongation factor EF-G shifts the mRNA one frame, expelling the uncharged tRNA

Elongation

Termination 2. A termination factor that recognizes the codon binds to the A site 3. The peptide in the P site is transferred to the factor 1. Translocation results in a stop codon (UAA, UGA, UAG) in the A site 4. Because the factor cannot bind, the peptide is released

Termination COOH

Diphtheria Toxin (Cornyebacterium diphtheria with 1. Reacts with elongation factor eEF-2 (prokaryote G) eEF-2 + NAD + bacteriophage cornyephage  ) + Break glycosidic bond to nicotinamide in reaction s s Trypsin s s A (catalytically active) B Diphtheria toxin (cell penetration) NAD + eEf-2 (diphthamide group) Modified His in eEF-2 is target ADP ribosylation

s s A B Receptor s s A B s H Endocytosis Disulfide cleavage s A NAD + eEF-2 ADP-ribosyl-eEF-2 Active enzyme H (ribosylates histidine residues with diphthamide structure) (See p 879)

Translation Blocking Antibiotics Puromycin Binds to A site and receives peptide from transpeptidase Aborts peptide prematurely Prokaryotes and eukaryotes

Aminoglycoside Causes misreading and inhibits chain initiation of prokaryote mRNA Blocks peptidyl transferase in prokaryotes (A-site binding) Inhibits aminoacyl-tRNA binding to prokaryotes: blocks nutritional factor ppGpp (p867)

Post-translational Processing Folding Proteolytic processing Additions to peptide chain Carbohydrates (glycosylation) Methyl groups Lipid groups Hydroxylation, deamination Secretion

Folding Rule: Proteins must fold to an active conformation. Generally, folding is spontaneous as the protein seeks its lowest energy level Rule: Cells have “chaperones” that assist in proper folding. Typical are Hsp70 and Hsp60

Random Chain Secondary helix or  sheet Secondary helix or  sheet Hydrophobic alignments Hydrophobic alignments Compact folded protein Compact folded protein Stepwise

Proteolytic Processing of Insulin A chain PreproinsulinProinsulinInsulin B chain Disulfide bonds s s s s ss s s