Exam #4: Protein Translation & Post-Translational Processing Flashcards
Eukaryotic Ribosomes
- Human Cytosolic vs. Mitochondrial
- 80S Assembled Size
- 60S Large Subunit w/ 50 Proteins, 25S, 5.8S & 5S RNA
- 40S Small Subunit w/ 34 Proteins. 18S RNA
Prokaryotic Ribosomes
- 70S Assembled Size (smaller)
- 50S Large Subunit w/ 34 proteins, 23S & 5S RNA
- 30S Small Subunit w/ 21 proteins, 16 S RNA
Differences between Prokaryotic & Eukaryotic Ribosomes
- Different assembled size
- Different size, # proteins, & RNA composition of small & large subunits
How are ribosomes assembled/ translation initiated?
- Small ribosomal subunit loaded with mRNA & tRNA
- The loaded small subunit binds the large subunit (concluding initiation)
- This process is guided by initiation factors
Outline Elongation
1) Initiator methionine-tRNA binds P site
2) Second aminoacyl-tRNA is placed into the A site, which is EF-1-GTP dependent (Elongation Factor-1)
3) Peptidyl bond is formed
4) Ribosome moves down one codon, which is EF-2-GTP dependent, and empties the A site
5) Uncharged tRNA leaves via the E site
How is translation terminated?
1) One of three STOP codons enters A site
2) Eukaryotic Release Factor (eRF)- GTP pairs w/ STOP codon
3) GTP is hydrolyzed & peptide is released from P site
4) Ribosome separates into subunits
How do chaperons assist in protein folding?
- Chaperones are proteins that associate with partly folded proteins
- Guide the folding process by binding hydrophobic regions
Where does exported protein synthesis occur?
- Synthesized on ER
- Trafficked through Golgi in vesicles
- Final destination
Unfolded Protein Response
Accumulation of unfolded proteins in the ER induces the unfolded protein response that:
- Inhibits protein translation
- Induces chaperone production
- Consider apoptosis if the amount of unfolded protein is in excess of repair
Glycosyltransferases
Transfer sugar form an activated sugar nucleotide to an acceptor substrate
What types of proteins are glycosylated and why?
- Cell surface & exported proteins are glycosylated
- B/c glycosylation increases solubility, stability, & size
- B/c glyocsylation puts a carbohydrate on the protein surface that is used as a recognition site
N-Linked Glycosylation
- Oligosaccharide added to amino group of asparagine
1) Synthesis of universal oligosaccharide on dolichol phosphate
2) Transfer of universal oligosaccharide to nascent polypeptide in ER
3) Modification of universal oligosaccharide in Golgi apparatus to produce high mannose and complex type
O-Linked Glycosylation
- Occurs only on fully folded protein in the Golgi apparatus
- Glycosyltransferases transfer N-acetyl-galatosamine to serine/ theronine
- Further transfer of sugars enlarges the carbohydrate
Differences between N-Linked & O-Linked Glycosylation
1) N-linked starts in ER & continues in Golgi; O-linked Golgi only
2) N-linked= asparagine, O-linked= serine/theronine
3) N-linked= universal first, then adjust; O-linked= add one carbohydrate at a time
Congenital Disorders of Glycosylation
Affect N-linked Glycosylation that impairs extracellular enzymatic functioning
- CDG-I= Defective synthesis of universal oligosaccharide
- CDG-II= Defective trimming of universal oligosaccharide
Post-translational Modificiation of Amino Acids
1) Proline hydroxylation (collagen)
2) Lysine acetylation (histones)
3) Thiol-group in cysteine converted into aldehyde to from C-alpha-formylglycine (lysosomal sulfatases)
4 Ways Hydrophobic Molecules are Added to Proteins
1) Myristoylation- addition of myrisitic acid to N-terminal
2) Palmitoylation- addition of palmitic acid to cysteine
3) Prenylation- addition of isoprenoids to cysteine near C-terminus
4) GPI anchor
Cystic Fibrosis
- Caused by a deletion of one gene from CFTR1
- Deletion interferes with protein folding & glycosylation
- Consequently, protein is degraded in cytoplasm instead of trafficking to plasma membrane
I-Cell Disease
- Transfer of phosphate to mannose is impaired
- Cannot generate mannose 6-phosphate for lysosomal degaradation
- Proteins accumulate in lysosome
Protein Import into Mitochondria
1) Mitochondrial proteins synthesized with N-terminal presequence
2) Chaperones stabilize in unfolded form
3) Presequence interacts with receptor in outer mitochondrial matrix
4) Complex of TOMs & TIMs provides a channel for the preprotein to enter the mitochondria
5) Presequence is cleaved by matrix proteases
Lysosomal Degradation
- Nonspecific degradation of extracellular & intracellular proteins
- High mannose glycoproteins are phosphorylated at mannose residues
- Mannose 6-Phosphate targets vesicles to lysosome
Proteasome Degradation
- Specific degradation of cytoplasmic proteins
- Signaled by poly-ubiquitination
Ubiquitin & Protein Degradation Steps
1) Activation= E1
2) Conjugation= E2
3) Ligation= E3
-Poly-ubiquitinated proteins are degraded
What factors determine protein half-life?
1) Conformation i.e. improperly folded are degraded
2) N-terminal residue
3) Other sequence elements e.g. PEST that shortens half life
