Translation Flashcards
mRNA is translated in the _ to _ direction and protein synthesis occurs from __ to __.
- mRNA translated in the 5’ to 3’ direction
- protein synthesis occurs from N-terminus to C-terminus
synonyms
codons that specify the same amino acid
degeneration of the codon
- amino acids specified by more than one codon
- one advantage of the genetic code: degeneracy of the codon minimizes effect of mutations
- many mutations at the third position are silent mutations
3 stop codons
- UAG (amber)
- UAA (ochre)
- UGA (opal)
What happens with mutations at the 2nd codon position?
- since purines (R) are all mostly polar and pyrimidines (Y) are all mostly hydrophobic, swapping an R for an R or a Y for a Y produces a smaller effect
- R–Y switch needed for a big change in amino acid
open reading frames (ORF)
- 3 possible reading frames for mRNA for a total of 6 possible reading frames for DNA
- only one reading frame will encode the protein
- AUG start codon indicates the beginning of ORF
frameshift vs. nonframeshift mutations
- frameshift: insertion or deletion of 1 or 2 bases; meaning of entire protein is lost
- nonframeshift: an entire codon is inserted or deleted; more similar to original and may have profound or minimal effects
tRNA (transfer RNA)
- at least one tRNA exists for every amino acid
- make up approx. 15% of the RNA pool
- average 80 nucleotides long
- 4 short segments fold into double helix, forming cloverleaf structure
- further folding into L shape held by H bonds
- have only 13 invariant nucleotides and 8 semi-invariant nucleotides
List the parts of the tRNA molecule beginning at the 5’ end.
- 5’ end
- acceptor stem
- D (dihydrouridine) stem and D loop
- anticodon stem, anticodon, anticodon loop
- variable loop
- T stem, T loop
- CCA attached to 3’ end
isoaccepting tRNAs
- tRNA recognizes multiple codons that specify the same amino acid
- e.g. GmAA carries Phe and can recognize both UUC and UUU
the wobble hypothesis
- pairing at position 1 and 2 are watson-crick but position 3 can be non watson-crick (wobble position)
- wobble position at third position (3’) of codon but first position (5’) on anticodon
- allows the same tRNA to recognize multiple codons that differ in only the wobble position
possible wobble anticodon (5’) - codon (3’) base pairs in bacteria
(anticodon base can pair with...) - A: U - C: G - G: C or U - U: A or G - I: A or C or U (in eukaryotes the only difference is that U only binds to A and I cannot bind to A)
tRNA charging
- attachment of the correct amino acid to the 3’ end of tRNA by aminoacyl-tRNA synthetase (aaRS)
- [amino acid + ATP] carboxyl group of amino acid linked to AMP
- [aminoacyl-AMP] adenylated amino acid linked to -OH on sugar of 3’ tRNA
- [aminoacyl-tRNA + AMP] amino acid and tRNA linked by ester linkage
aminoacyl-tRNA synthetases (aaRS)
- enzymes involved in tRNA charging
- activation of amino acid for protein synthesis
- class I aminoacylates at the 2’–OH
- class II aminoacylates at the 3’–OH
aaRS editing
- editing capabilities in case of mistakes
- after the amino acid is linked to AMP, aaRS forces it from synthesis site into its second pocket (editing site)
- excludes correct amino acid
- keeps closely related but incorrect amino acid and removes it from AMP/tRNA
- increases tRNA charging accuracy
prokaryotic ribosomes (70S)
- composed of 50S large subunit and 30S small subunit
- 50S: 23S and 5S rRNA + 33 proteins
- 30S: 16S rRNA + 21 proteins
eukaryotic ribosomes (80S)
- composed of 60S large subunit and 40S small subunit
- 60S: 5S, 5.8S and 28S rRNA + 49 proteins
- 40S: 18S rRNA + 33 proteins
small ribosomal subunit vs. large subunit function
- small subunit: channel threads mRNA, matches tRNAs to codon
- large subunit: channel for growing polypeptide chain, catalyzes peptide bond formation
16S rRNA
- structural role, scaffold for protein binding
- 3’ end: translation initiation + S1 & S2 proteins
- 30S-50S interaction + tRNA interaction in A&P sites
- stabilizes correct codon-anticodon pairing
23S rRNA
- structural role, scaffold for protein binding
- 30S-50S interaction + tRNA interaction in A&P sites
- proton acceptor during peptidyl transferase reaction
- a ribozyme: catalyzes peptide bond formation
Shine Dalgarno sequence
- bacterial mRNA translation
- base pairs with 3’ end of 16S rRNA at anti-shine dalgarno sequence (CCU CCU)
- upstream of AUG start site at -16 position
- 3-10 nucleotides long
- appears in front of every translation start site (polycistronic)
initiation in prokaryotes
1) ribosome reactivation
- IF-1 and IF-3 bind to the 30S subunit and block the A site
2) formation of 30S initiation complex
- IF-2 + GTP binds
- Shine Dalgarno on mRNA interacts with anti-Shine Dalgarno on 16S rRNA of 30S subunit
- fMet-tRNAfmet docks to P site and binds to AUG start codon of mRNA
3) formation of 70S initiation complex
- binding of fMet-tRNAfmet triggers release of IF-1 and IF-3
- IF-2 hydrolyzes GTP into GDP + Pi
- 50S subunit joins
elongation in prokaryotes
1) binding of substrate
- aa-tRNAaa + EF-Tu + GTP complex binds to A site
- if amino acid is correct, EF-Tu will hydrolyze GTP to GDP and release tRNA
2) transpeptidation
- amino acid at P site transferred to amino acid at A site, catalyzed by 23S rRNA (peptidyl transferase ribozyme)
3) translocation/ratcheting
- 30S subunit turns 7-8 degrees clockwise, moving mRNA and tRNA
- tRNA moves 1 full site in 50S subunit and 1/2 site in 30S subunit
- GTP - EF-G power stroke moves 30S tRNA the other 1/2 site
- 30S subunit returns into original position
termination in prokaryotes
Phase 1:
- stop codon (UAG, UAA, or UGA) enters A site
- RF-1 binds to UAG or UAA, RF-2 bins to UGA or UAA
- RF-3 comes in with GDP, GDP dissociates from RF-3 and GTP takes its place
- hydrolysis of GTP causes RF-1, RF-2 and RF-3 to dissociate
Phase 2:
- peptidyl transferase transfers polypeptide to H2O instead of amino acid
- peptide is released from ribosome
Phase 3:
- tRNA and RFs dissociate, mRNA released (GTP driven)
