Post on 20-Dec-2015
TranslationThe participants
Ribosome - ribonucleoprotein enzyme and structural complex
mRNA - carries the DNA information encoding the protein
tRNAs - decoders of mRNA
amino acids and N-formylmethionine
Translation initiation
Initiation complex formation
(30S ribosome subunit-mRNA-fMet-tRNA)
Elongation
Binding charged tRNA
RNA in large ribosomal subunit is the enzyme in peptide bond formation
Translocation
Termination
Stop codons
Release factors
A comparison of the structures of procaryotic and eucaryotic ribosomes.
The ribosome is an allosteric enzyme.The peptidyl-transferase site is the ACTIVE SITE
The Prokaryotic Cast (in eukaryotes similar but more complex)
Ribosome ~3 x 106 Da, 250 Å (50S + 30S = 70S)The ribosome provides the structure necessary for translation
and catalyzes the reaction
What else is needed?
Factors:
• IF 1, 2,3 Initiation
• EF-Tu, EF-Ts, EF-G Elongation
• RF 1, 2, 3, RRF Release
• GTP hydrolysis
Figure 32-43Some translational initiation (Shine-Dalgarno) sequences recognized by E. coli ribosomes.
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Shine-Dalgarno sequences typically start 10-15 nt upstream of the initiation codon.
Are only found in prokaryotes.
Figure 32-45Translational initiation pathway in E. coli.
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• 50S and 30S associated.
• IF3 binds to 30S, causes release of 50S.
• mRNA, IF2-GTP (ternary complex), fMet-tRNA and IF1 bind 30S.
• IF1 and IF2 are released followed by binding of 50S.
• IF2 hydrolyzes GTP and poises fMet tRNA in the P site.
Defining tRNA Binding Sites in Different functional States
GENERATE RIBOSOMES IN THE FOLLOWING STATES:
A (Aminoacyl): EF-Tu.GTP dependent; mRNA dependent; occupied P Site
P (Peptidyl): Reactive with Puromycin (Pm)E (Exit): Deacylated tRNA
MONITOR BY CHEMICAL FOOTPRINTING: 30S A site protections:50S P site protections
(also X-linkers, EDTA-FeII) Looking at footprint pre and post peptide bond, translocation
The data didn't fit into a simple 2 site modelHYBRID STATES HAD TO BE INVOKED
tRNA movement occurs independently on 2 subunits via 6 hybrid states.
1. A/T --> 2. A/A --> 3. A/P --> 4. P/P --> 5. P/E --> 6. E
In this model the tRNA would "ratchet" its way through the ribosome undergoing 50° rotations along its longitudinal axis from A to P.
This model has received support from EM and X-ray studies.
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RF-1 = UAARF-2 = UAA and UGA
Cannot bind if EF-G is present.
RF-3-GTP binds to RF1 after the release of the polypeptide.
Hydrolysis of GTP on RF-3 facilitates the release of RF-1 (or RF-2).
EF-G-GTP and ribosomal recycling factor (RRF)-bind to A site. Release of GDP-RF-3
EF-G hydrolyzes GTP -RRF moves to the P site to displace the tRNA.
RRF and EF-G-GDP are released yielding inactive 70S