Ribosome Structure eukaryotes small subunit (40S) synthesized in the nucleus large subunit (60S) synthesized in the nucleolus total = 80S S = sedimentation prokaryotes small subunit (30S) + large subunit (50S) → 70S Catalytic sites A (acceptor) site accepts charged aa-tRNA P (peptidyl) site holds the growing peptide chain and transfers to the aa-tRNA in the A site E (exit) site allows tRNA to exit the ribosome after releasing its amino acid to the aa-peptide chain Forms polysomes single mRNA being translated simultaneously by several ribosomes can be free proteins for nucleus or mitochondria membrane bound found on rough ER proteins for secretion or membrane insertion Steps Initiation initiation complex generated from small ribosomal subunit mRNA 5'-cap (eukaryotes) Shine-Dalgarno sequence (prokaryotes) initiation aa-tRNA met-tRNA (eukaryotes) fmet-tRNA (prokaryotes) initiation factors (IFs) help in assembly released when mRNA joins the ribosomal subunit once assembled, recruits the large ribosomal subunit initiation aa-tRNA occupies the P-site all other aa-tRNAs bind the A-site the A-site is empty Elongation aminoacyl-tRNA binds to A site consumes 1 GTP peptidyl-bond formation peptidyl transferase reaction catalyzed by ribosomal rRNA ("ribozyme") transfers growing polypeptide to amino acid in the A site translocation ribosome moves 3 nucleotides at a time in a 5' to 3' direction moves peptidyl RNA from A site to P site moves empty tRNA from P site to E site for exit A site now empty and ready to accept next aa-tRNA catalyzed by elongation factor-2 (eEF-2) inactivated via ADP-ribosylation by bacterial toxins (Pseudomonas and Diphtheria) consumes 1 GTP cycle is repeated for each amino acid addition to the polypeptide Wobble phenomenon The anticodon of certain tRNA molecules can bind to multiple codons The first two nucleotides of the codon adequately specify which tRNA (and amino acid) binds the codon, thereby permitting multiple nucleotides in the third position to bind the same tRNA. Termination stop codon reached in mRNA release factor binds mRNA peptide bond is hydrolyzed and completed protein is released ribosomal complex dissociates Overall energy requirement 4 high energy bonds ATP to AMP for tRNA charging (2) 2 GTP to GDP for elongation (2) Pharmacologic Inhibition of Prokaryotic Protein Synthesis Bind 30S aminoglycosides inhibit formation of the initiation complex promotes misreading of mRNA Bind 50S chloramphenicol inhibits peptidyl transferase macrolides inhibits translocation clindamycin inhibits translocation