Ribosomes
large complexes of proteins and rRNA.
subunits assemble together into active complex in the presence of mRNA
3 sites:
A - Acceptor site (mRNA codon exposed to receive aminoacyl tRNA, except methionine tRNA)
P - Peptidyl site (where aminoacyl tRNA is attached)
E - Empty or Exit site (location occupied by empty tRNA before exciting ribosome)
streptomycin
inhibits initiation step (prok)
binds to 30S subunit
Tetracycline
inhibit elongation step (prok)
binds to 30s subunit
Shiga toxin
inhibit elongation step (euk)
binds to 60S to disrupt elongation
Ricin toxin
inhibits elongation step (euk)
binds 60S subunit and blocks entry of aminoacyl tRNA to ribosome complex
puromycin
inhibits elongation step (prok/euk)
- resembles 3’ end of aminoacylated tRNA and enters A site so no new AA can enter
used in the LAB
Chloramphenicol
inhibits elongation step (prok/mit)
inhibits peptidyl transferase
Cyclohexamide
inhibits elongation step (euk)
inhibits peptidyl transferase and impairs peptide bond formation
Clindamycin
inhibits elongation step (prok)
bind to 50s Subunit and disrupt translocation
Diptheria Toxin
inhibits elongation step (euk)
inactivates EF2-GTP
Protein sorting - cytoplasmic pathway
proteins destined for cytosol, mitochondria, nucleus, and peroxisomes
initial synthesis begins AND ends on free ribosomes in cytosol
Protein sorting - secretory pathway
proteins destined for ER, lysosomes, plasma membrane, or secretion
- TSN begins on free ribosomes but terminates on ribosomes sent to ER
- proteins have ER targeting signal sequence on 1st 20 AA on polypeptide
- all proteins go through ER lumen for quality control, then to golgi apparatus
- SRP binds ER target signal and hugs ribosome during translation, then tethers it to ER membrane and temporarily halts translation
- translation continues and protein is fed through protein translocator into ER lumen, enzymes on luminal side cleave protein
cytoplasm protein signal
no signal
cytoplasmic pathway
mitochondrial protein signal
a-helix signal made of N-terminal hydrophobic a-helix
cytoplasmic pathway
proteins destined for nucleus
Lysine and Arginine rich
cytoplasmic pathway
peroxisome protein signal
sequences rich in serine, lysine, and leucine (SKL)
cytoplasmic pathway
ER protein signal
KDEL (lysine, asparagine, Glutamine, leucine)
- secretory pathway
Plasma membrane signal
N-terminal apolar residues
- secretory pathway
Lysosome protein signal
Mannose 6-Phosphate
- secretory pathway
Secreted protein signal
Trp rich domain
- secretory pathway
- put into vesicles and fuse with the PM
Mitochondrial protein import
proteins meant for specific place in mitochondria
- unfolded proteins protected by HSP 70 (chaperones so they don’t get degraded)
- two channels: (TOM on Route membrane and TIM on inner membrane)
- pores are so small that proteins have to be unfolded to get in
nuclear protein import
- nuclear pores are large enough for small folded proteins to enter
- large proteins need nuclear localization signal (four continuous basic residues)
Inclusion Cell disease
- secretory pathway disease
- lysosomal proteins not tagged with M6P because they have defective or missing GlicNAc phosphotransferase
- proteins are not phosphorylated, therefore not sorted into vesicles and not transferred to lysosome
- instead carried to surface and secreted (found in blood)
Protein folding
large proteins need help folding, can’t risk aggregation or proteolysis
chaperone proteins - HSP70
chaperonin proteins - HSP60 (barrel shaped), fold in ATP dependent manner
