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)
initiation in eukaryotes (difference from prokaryotes)
- instead of Shine Dalgarno, the 5’ cap along with eIF4E&4G with other eIFs form the 43S initiation complex
- initiator tRNA moves along mRNA searching for AUG start codon and stops at Kozak sequence at which point various eIFs are released and large subunit binds
List the prokaryotic vs. eukaryotic component that:
- binds aminoacyl-tRNA
- promotes translocation
- recycling of EFs
- binds aminoacyl-tRNA: EF-Tu in prokaryotes and eEF-1 in eukaryotes
- promotes translocation: EF-G in prokaryotes and eEF-2 in eukaryotes
- recycling of EFs: EF-Ts in prokaryotes and eEF-1 in eukaryotes
prokaryotic vs. eukaryotic release factors
- prokaryotes: RF-1, RF-2 and RF-3
- eukaryotes have only one RF
The 30S initiation complex is composed of…
30S subunit + IF-1 + IF-2 + IF-3 +mRNA + fMet-tRNAfMet + GTP
Which translation factors bind to GTP?
- IF-2
- EF-Tu
- EF-G
- RF-3
Post translational modifications
- cleavage of initiator Met
- cleavage of signal sequences
- addition of cofactors
- glycosylation, acetylation, phosphorylation
- multi subunit proteins assemble
- transportation to site of action
ubiquitin cycle
- start with monomeric ubiquitin
- activate it (E1)
- conjugate together and add bad protein (E2)
- use 26S proteosome and ATP to release short peptide fragments and polyubiquitin chain
- polyubiquitin chain broken into monomeric ubiquitin
