Zaidi, Protein Processing Flashcards
Start Codon
AUG, Methionine
Stop Codons
UAA, UAG, UGA
Silent Mutation
new codon, no change in AA, no effect on protein
Missense Mutation
new codon, a change in the AA, variable effect
if a similar AA: no effect on protein
if a different AA: changes protein effect (sickle cell anemia)
Nonsense Mutation
new codon, changes into a stop codon, nonfunctional protein
Frameshift Mutation
1+ nucleotide added/deleted, creates nonfunctional protein (duchenne muscular dystrophy)
Sickle Cell Anemia
missense mutation of 6th codon in allele for human beta global (HBB)
changes Glu into a Val (go from negatively charged and hydrophilic to neutral and hydrophobic)
HbA aggregates in rod-like structure, deforms RBC’s, poor oxygen capacity and clog capillaries
Duchenne Muscular Dystrophy
deletions of dystrophin gene leads to partial or non-functioning dystrophin protein
Frameshift mutation
Out of frame (OOF) causes little to no expression of protein and cause severe form of DMD
in frame results in truncated forms of protein and is milder (Becker MD)
sx: muscle wasting, death by resp. failure by 10yo
mRNA
has codons for AA
after transcription pre-mRNA into mRNA and then goes to cytoplasm for translation
7-methylguanosine (5’ cap) & Poly(A) tail
tRNA
adaptors, transfer AA to ribosome for synthesis
match AA to codons in mRNA
anticodon loop- pair with complementary codon
3’CCA terminal region- binds the AA that matches
Codons
sequence of 3 bases that signal for a protein
64 possibilities, 3 stop codons, 1 start codon,
61 triplet codons for 20 AAs
degenerate- some AA have multiple sequences
Aminoacyl tRNA synthetase
catalyzes addition of AMP to COOH to activate the AA
each AA has its own aminoacyl tRNA synthetase
Ribosomes
translation occurs here!
large and small subunit (eukary- 30s & 50s, prokary- 40s & 60s)
Acceptor site (A)- mRNA codon exposed to receive aminoacyl tRNA, new tRNA comes in
Peptidyle site (P)- aminoacyl tRNA attaches here
Empty/Exit site (E)- empty tRNA before exiting ribosome,
Translation
•Translation occurs in the 5’ to 3’ direction
3 steps:
1. Initiation: formation of mRNA, small ribosomal subunit and initiator tRNA pre-initiation complex
2. Elongation: activated AA attached to initiating Met by forming a peptide bond
3. Termination: peptide chain released
Translation, Initiation
Protein synthesis begins
requires 1 GTP
Initiator tRNA = methionyl tRNA, which is bound to GTP is attached to P site on the Small su
other eIF’s are added
the Large su is added once the eIF’s and GTP fall off and are removed
translation begins with the initiation codon AUG (code for Met)
eIF’s
eukaryotic initiation factors
**see this, think translation initiation
Translation, Elongation
Requires 2 GTP for every AA attached
Aminoacyl tRNA (attached to GTP bound elongation factor-EF) binds with next AA at the A site
2 AA (Met and next one) attached via peptide bond catalyzed by peptide transferase
continued cycle
Translation, Termination
Peptide chain released and ribosomal complex dissociates
requires 1 GTP
triggered by stop codons (UGA, UAA, UAG)
stop codons recognized by Release Factors (RFs) which bind to the A site, and cleave the ester bond between C-terminus and the tRNA
GTP hydrolysis dissociates ribosome complex
Inhibitors of Translation
Streptomycin-disrupts initiation, bind to 30s
Clindamycin; erythromycin- disrupt translocation and thus elongation, bind to 50s
Tetracyclines- disrupt elongation, bind 30s
More elongation disruptors:
- shiga toxin-bind 60s
- ricin
- puromycin
- diptheria toxin- bind EF2-GTP
- cycloheximide
- chloramphenicol
Cytoplasmic pathway
proteins destined for:
- cytoplasm
- mitochondria: N terminal hydrophobic alpha-helix
- nucleus: KKKRK
- peroxisome: SKL
Secretory pathway
protein sorting, trafficked via vesicles
- membrane: N terminal apolar (stop tsrf)
- lysosome: Mannose 6-P
- secretory vesicle: Trp rich domain
- stay in ER: KDEL
Mitochondria transport & Cytoplasmic Pathway
proteins get help from chaperone proteins (heat shock family, hsp) which protect the protein in it’s linear form as it passes through membrane
TOM-Transporter in outer membrane
TIM- Transporter in inner membrane
chaperone: HSP70
Signal recognition particle (SRP)
- binds to ER-targeting signal, wraps around ribosome-mRNA-peptide complex and halts translation
- binds to SRP receptor on ER membrane
- resumes translation when protein is put into the ER lumen
- enzymes inside of membrane cleave signal (SRP)
Post-translational processing; protein folding
- small proteins have no problem
- large proteins are at risk for damage and thus need chaperons and chaperonins (use ATP)
- chaperone = HSP70 -chaperonin = HSP60
