8. Protein Synthesis and Protein Processing a). Ribosome structure b). Protein synthesis i). Initiation of protein synthesis ii). Peptide bond formation;

8. Protein Synthesis and Protein Processing a). Ribosome structure b). Protein synthesis i). Initiation of protein synthesis ii). Peptide bond formation; peptidyl transferase iii). Elongation and termination iv). Inhibitors of protein synthesis Antiviral action of interferon Induction of 2-5A synthase Induction of eIF 2 kinase Antibiotics c). Protein processing i). Synthesis of secreted and integral membrane proteins ii). Glycosylation and protein targeting iii). Proteolytic processing

C NH 2 CH 3 -S-CH 2 -CH 2 -CH O=C Peptide bond formation peptide bond formation is catalyzed by peptidyl transferase peptidyl transferase is contained within a sequence of 23S rRNA in the prokaryotic large ribosomal subunit; therefore, it is probably within the 28S rRNA in eukaryotes the energy for peptide bond formation comes from the ATP used in tRNA charging peptide bond formation results in a shift of the nascent peptide from the P-site to the A-site NH 2 CH 3 -S-CH 2 -CH 2 -CH O=C O tRNA NH 2 CH 3 -CH O=C O tRNA N P-site A-site OH tRNA NH CH 3 -CH O=C O tRNA

Induction and action of interferon virus virus invades cell cell makes interferon in response to viral RNA cell cannot protect itself virus replicates cell succumbs interferon binds to receptors on neighboring cells and activates the cells cell synthesizes antiviral proteins in response to interferon activation virus invades neighboring cell cell protected from viral infection by antiviral proteins

Functions of two antiviral proteins interferon induces ATP viral dsRNA 2-5A synthase oligo 2-5 adenylate (2-5A) [-A-2’-p-5’-A-2’-p-5’A-] N eIF2 viral dsRNA eIF2 kinase eIF2 P activeinactive: viral protein synthesis cannot initiate inactive endonuclease active endonuclease: viral mRNA degraded

Inhibitors of protein synthesis Inhibitor Process Affected Site of Action Kasugamycin initiator tRNA binding30S subunit Streptomycin initiation, elongation30S subunit Tetracycline aminoacyl tRNA bindingA-site Erythromycin peptidyl transferase50S subunit Lincomycin peptidyl transferase50S subunit Clindamycin peptidyl transferase50S subunit Chloramphenicol peptidyl transferase50S subunit Staphylococcus resistance to erythromycin certain strains of Staphylococcus can carry a plasmid that encodes an RNA methylase this RNA methylase converts a single adenosine residue in 23S rRNA to N 6 -dimethyladenosine this is the site of action of erythromycin, lincomycin, and clindamycin N 6 -dimethyladenosine blocks the action of these antibiotics the organism that produces erythromycin has its own RNA methylase and thus is resistent to the antibiotic it makes

Protein maturation: modification, secretion, targeting 5’ AUG polysome for secreted protein 2. the signal recognition particle a (SRP) binds the signal peptide b and halts translation 1. translation initiates as usual on a cytosolic mRNA a the signal recognition particle (SRP) consists of protein and RNA (7SL RNA); it binds to the signal peptide, to the ribosome, and to the SRP receptor on the ER membrane b the signal peptide is a polypeptide extension of 10-40 residues, usually at the N-terminus of a protein, that consists mostly of hydrophobic amino acids c ER = endoplasmic reticulum ER lumen c cytosol 3. the SRP docks with the SRP receptor on the cytosolic side of the ER membrane and positions the signal peptide for insertion through a pore SRP SRP receptor Translation of a secreted protein

5’ ER lumen cytosol 4. translation resumes and the nascent polypeptide moves into the ER lumen 5. signal peptidase, which is in the ER lumen, cleaves off the signal peptide 7. the ribosomes dock onto the ER membrane; the rough ER is ER studded with polysomes 6. the SRP is released and is recycled

5’ ER lumen cytosol UGA 8. translation continues with the nascent polypeptide emerging into the ER lumen 9. at termination of translation, the completed protein is within the ER and is further processed prior to secretion completed protein is processed and secreted

Examples of secreted proteins: polypeptide hormones (e.g., insulin) albumin collagen immunoglobulins Integral membrane proteins are also synthesized by the same mechanisms; they may be considered “partially secreted” Examples of integral membrane proteins: polypeptide hormone receptors (e.g., insulin receptor) transport proteins ion channels cytoskeletal anchoring proteins (e.g., band 3)

Glycosylation of proteins most integral membrane proteins and secreted proteins are glycosylated during translation on the ER membrane the protein begins to be glycosylated various oligosaccharide modifications occur in the ER and in the Golgi complex O-linked (Ser, Thr linked) oligosaccharides (linked to hydroxyl group) N-linked (Asn linked) oligosaccharides (linked to amide group) Biosynthesis of N-linked oligosaccharides (first 7 steps) ER lumen Cytosol P (1) UMP, (1) UDP Dolichol phosphate (polyprenol lipid carrier) N-acetylglucosamine (GlcNAc) = Mannose = (2) UDP- P P (5) GDP- (5) GDP P P reorientation Monosaccharides are transferred by specific glycosyltransferases from nucleotide sugars

PP ER lumen Dolicol-phosphates are the sugar donors in the ER lumen; they are synthesized in the cytosol prior to being translocated to the lumen Cytosol PP (4) (3) Dolicol-P-mannose = Dolicol-P-glucose = P P P P Biosynthesis of N-linked oligosaccharides (second 7 steps)

ER lumen Cytosol PP Linkage is to the amide group of an asparagine followed by any (X) amino acid (except proline) followed by serine or threonine Transfer of oligosaccharide chain to the growing polypeptide Asn I X I Ser (Thr) Following synthesis, the protein is transferred to the Golgi complex, where trimming and further building of the oligosaccharides occurs Transfer of oligosaccharide to protein

Asn I X I Ser (Thr) Asn I X I Ser (Thr) Trimming by glycosidases ; Building by glycosyltransferases A complex type oligosaccharide fucose = galactose = sialic acid = come from nucleotide sugars translocated across the Golgi membrane Golgi lumen Cytosol The type of carbohydrate determines whether the protein is targeted to the membrane, to a vesicle, or is secreted = common core structure Formation of complex type oligosaccharides

Targeting of proteins to lysosomes (I-cell disease) Asn UDP- P P Asn P P Proteins containing mannose-6-phosphate are targeted to lysosomes Patients with I-cell (for inclusion body) disease have a deficiency in the enzyme that transfers GlcNAc phosphate to mannose residues in the Golgi Phosphate groups are added to mannose by transfer of GlcNAc phosphate from UDP-GlcNAc The resulting deficiency in lysosomal hydrolases results in an accumulation (inclusions) of material in the lysosomes These proteins include the lysosomal hydrolases As a result, the hydrolases cannot be targeted to the lysosomes

Proteolytic processing Processing of insulin (synthesized in the ER of pancreatic  -cells) N C Preproinsulin cleavage of signal peptide by signal peptidase Signal peptide C SISSIS SISSIS N Proinsulin C SISSIS SISSIS N C-chain Cleavage by trypsin-like enzymes releases the C-peptide C SISSIS SISSIS N Insulin C N Disulfide bond formation Further trimming by a carboxypeptidase B-like enzyme removes two basic residues from each of the new ends C-chain The C-chain is packaged in the secretory vesicle and is secreted along with active insulin B-chain A-chain

Preproopiomelanocortin multiple functional polypeptides from a single precursor processed in a cell-specific manner 26aa 48aa 12aa 40aa 14aa 21aa 40aa 18aa 26aa N C Signal peptide Proopiomelanocortin Corticotropin (ACTH)  -MSH  -Lipotropin  -MSH  -MSH Endorphin  -Lipotropin Enkephalin (5aa) 31aa 5aa