FCH 532 Lecture 19
This material will not be on the exam
Chapter 32: Translation
Translation
Ribosome - ribonucleoprotein enzyme and structural complex
The participants
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
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Figure 32-41 Ribosomal peptidyl transferase reaction
forming a peptide bond.
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Figure 32-43 Some translational initiation (ShineDalgarno) sequences recognized by E. coli ribosomes.
Shine-Dalgarno sequences typically start 10-15 nt upstream
of the initiation codon.
Are only found in prokaryotes.
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Figure 32-45 Translational
initiation pathway in E. coli.
• 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.
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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 model
HYBRID 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.
cryo-EM
Aminoacyl-tRNA
EF-Tu-GTP
EF-Ts
GTP
EF-Tu-EF-Ts
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EF-Ts
GDP
RF-1 = UAA
RF-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).
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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
Translation
•
•
•
•
Shine-Dalgarno sequence
Initiation
Elongation
Release
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