Lecture 38: Protein Synthesis I Flashcards
Components of protein synthesis process
- mRNA
- Ribosomes (organelle)
- tRNA (adaptor)
- Genetic code (language)
Features of genetic code in protein synthesis
- Degenerate (more than 1 codon for some AAs)
- Not ambiguous (no shared codons)
- Almost universal (besides mitochondria)
Mutation types
- Point
- Silent
- Missense
- Nonsense
- Insertion/deletion (frameshift)
Point mutation
Single base change
Silent mutation
Mutation results in same AA produced (codon degeneracy)
Missense mutation
Mutation results in different AA produced
Nonsense mutation
Mutation results in new stop codon
Insertion/deletion mutation (frameshift)
Adding or deleting 1 or more bases; frameshift if not multiple of 3
tRNA anti-codon/codon binding specificity and wobble
5’ and middle base in anti-codon/codon (tRNA/mRNA) interaction must bind perfectly
3’ base has wobble; flexible binding
Factor types needed for protein synthesis
- I (initiation)
- E (elongation)
- R (release)
AA activation steps
Both steps catalyzed by aminoacyl tRNA synthetase (specific kinds for given AAs)
1. AA + ATP → aminoacyl adenylate:synthetase + PPi
2. Aminoacyl adenylate:synthetase + tRNA → AA-tRNA + AMP + PPi
Role of AA activation for protein synthesis
Controls protein synthesis accuracy
1. Each synthetase has to recognize the AA and correct tRNA seq.
2. Each synthetase has activation site + hydrolytic site (error correction)
3. AA-tRNA that leaves cannot be corrected further
fMet
Formyl group added to Met w/ initiator tRNA to form fMet; all proteins start w/ fMet
Ribosome tRNA-AA sites
- A site (aminoacyl)
- P site (peptidyl)
- E site (exit)
Shine-Dalgarno sequence
At 5’ end of prokary. mRNA. Sets reading frame; 1st codon is always AUG (fMet) after Shine-Dalgarno.
Prokaryotic translation initiation process
- Shine-Dalgarno positions 30S subunit
- Initiator tRNA bound to fMet + IF2-GTP is positioned in P site
- 50S subunit binds; IF2-GDP + other IFs release
Thus formation of 70S initiation complex
Translation elongation cycle steps
- EF-Tu-GTP mediates binding codon specific AAcyl-tRNA to A site
- Peptide bond formation between growing peptide chain/tRNA in A site; mediated by peptidyl transferase ribozyme
- Free tRNA left in P site
- Ribosome translocation w/ EF-G-GTP opens new free A site for next tRNA
Translation termination process
- Stop codon appears in A site
- RF1/2 bind A site
- RF3 binds common site
- Peptidyl transferase ribozyme catalyzes ester bond cleavage w/ GTP hydrolysis to release protein, tRNA, mRNA, ribosomal subunits
Ribosome common site
EF-TU, EF-G, RF3 all bind same “common” site. This ensures 2 processes can’t occur simultaneously.
Polyribosomes
Prokaryotic + eukaryotic feature where many ribosomes translate a gene simultaneously
Protein synthesis inhibitors
- Tetracycline
- Chloramphenicol
- Puromycin
Tetracycline
Blocks AAcyl-tRNA binding w/ A site in prokaryotes
Chloramphenicol
Resembles peptide bond, inhibiting peptidyl transferase in prokaryotes
Puromycin
Enters A site and accepts polypeptide chain, blocking translocation of ribosome in prokary. + eukaryotes.
Energy use for 100 AA peptide
- AA activation cleaves 2 high energy bonds
- Initiation already places first tRNA in P site
- Subsequent tRNAs need energy for A site binding + translocation (2 bonds)
- Termination uses 1 bond
Hep C virus
Flavivirus; HCV virus + strand RNA use host ribosomes to synthesize viral protein. Does NOT insert into DNA
HIV
Inserts into genetic material and uses host genetic material to replicate
CF mutations
E.g. early stop codon (W1282X) causes premature stop
