Protein synthesis II
Biochemistry 302
Bob Kelm
February 23, 2005
Several idealized views of the 70S
ribosomal complex during translation
70S cavity
50S “tunnel”
Fig. 27.25
View with 30S subunit in
front, 50S subunit behind
Both models imagine all three
binding sites (A, P, E) occupied
by tRNAs. This would only be a
transient occurrence during
actual protein synthesis.
Prokaryotic translation: Three steps of
chain elongation
• A site (AA-tRNA binding, EF-Tu-GTP hydrolysis)
– Loading of new AA-tRNA joined to EF-Tu-GTP
– Codon positioning of AA-tRNA assisted by GTP hydrolysis
– Dissociation of EF-Tu-GDP (Released EF-Tu “reloaded” with
GTP via EF-Ts exchange factor)
• A,P sites (transpeptidation in 50S subunit)
– α-amino group from A site AA-tRNA attacks the carbonyl
carbon of P-site bound peptidyl-tRNA
– Formation of new peptide bond at A/P 50S hybrid-site
– P-site deacylated tRNA (i.e. w/o peptide) - leaving group
• A, P, E site (translocation, EF-G-GTP hydrolysis)
– Rapid transfer of uncharged tRNA to E site and ejection
– Translocation of peptidyl-(3′OH) tRNA from A site to P site via
EF-G-mediated ribosome movement 3′ to the next codon
Stage 3: Elongation Step 1: Binding of
second aminoacyl-tRNA (EF-Tu-GTP)
Proofreading of codon
anticodon interaction likely
occurs at this step to allow
“incorrect” aminoacyl-tRNAs
to dissociate.
Regeneration of EF-TuGTP: This “reloading”
cycle does not involve
any GTP hydrolysis.
Regeneration occurs
by EF-Ts mediated
nucleotide exchange.
Lehninger Principles of Biochemistry, 4th ed., Ch 27
Stage 3: Elongation Step 2: Formation
of first peptide bond (“mobile” tRNAs)
α
Structural constraints
necessitate that both
tRNAs likely shift their
position in the 50S
subunit to assume a
hybrid binding states.
There is no E site tRNA
anticodon binding
domain in 30S subunit.
Large 50S subunit:
Peptidyltransferase
ribozyme complex
Small 30S subunit:
Proofreading occurs
after the charged tRNA
is in place and both
before and after GTP
hydrolysis by EF-Tu.
Lehninger Principles of Biochemistry, 4th ed., Ch 27
Stage 3: Elongation Step 3: Ribosome
translocation to next codon
Facilitates change
in ribosome
conformation
EF-Tu + tRNA
EF-G + GDP
…ready for next cycle of elongation. Note how functional
connection between mRNA template and the decoded
polypeptide product is maintained.
Peptidyl transfer and translocation
likely involves hybrid ribosome states
(an idea championed by Harry Noller)
EF-Tu: GDP
Proofreading (ms
time scale)
3-nt step
Chemistry
can happen
here.
A look at the putative transition state
of peptidyl transferase
P site
A site
Adenosine
3′
α
Tetrahedral
carbon
intermediate
resolves to yield
a deacylated
tRNA (P) and a
peptidyl tRNA
extended by one
amino acid.
P. Nissen et al. Science 289:920-929, 2000
Peptidyl
transferase
inhibitors with
P or A site
ribosome
binding sites.
Puromycin
resembles 3′
end of aminoacylated tRNA.
How puromycin inhibits protein synthesis
Puromycin is made by the mold
Streptomyces alboniger and
affects both prokaryotic and
eukaryotic ribosomes.
Structure of peptidyl-puromycin
Ribosome-specific antibiotic inhibitors
•
Cycloheximide
– Affects 80S ribosome only
– Blocks peptidyl transfer
•
Streptomycin
– Bacterial 30S subunit specific
– Causes codon misreading
•
Tetracycline
– Bacterial A site drug
– Block AA-tRNA binding (can’t
pass thru euk cell membranes)
•
Chloramphenicol
– Affects bacterial, mitochondrial,
chloroplast ribosomes only
– Blocks peptidyl transfer
•
Erythromycin
– Binds bacterial 50S subunit
– Blocks elongation/translocation
Atomic view of peptidyl transferase
region of Haloarcula marismortui
No proteins near
(∼18 angstroms)
of active site.
Catalytic activity
depends entirely
on RNA.
Atoms belonging
to 23S rRNA >95%
conserved in all
three kingdoms
are red.
P. Nissen et al. Science 289:920-929, 2000
Catalytic potential achieved by making
A2486 a stronger base via charge relay
Negative electrostatic
charge originating
from buried A2485
phosphate could be
relayed to N3 of A2486
via the proposed
mechanism to
generate an imino
tautomer.
Charge relay
mechanism is
important in
serine protease
catalysis.
N3 of A2486 is
~3 Å from
phosphoramide
oxygen and 4 Å
from amide N.
imino
3
2
P. Nissen et al. Science 289:920-929, 2000
Raising the pKa of
A2486 makes the
proximal α amino
group of AA-tRNA a
better nucleophile
N3 represented as
standard tautomer but
is thought to function
as a general base.
3
P site
A site
3
Tetrahedral carbon
intermediate stabilized
by H-bonding between
protonated N3 imine
group and oxyanion.
3
P. Nissen et al. Science 289:920-929, 2000
Deacylation: Proton
transfer from N3 to the
peptidyl-tRNA 3′OH.
Stage 4:Termination of polypeptide
synthesis
•
•
Signaled by ribosome encountering
a stop codon in the A site
No corresponding stop tRNA (except
for Sec-tRNASec) so release factor
complex binds to ribosome instead.
– RF-1 (UAG, UAA)
– RF-2 (UGA, UAA)
– RF-3 (GTPase, needed to release 50S
ribosome subunit)
•
•
•
Peptidyltransferase transfers P-site
peptide chain to a water molecule.
Unstable 70S ribosome dissociates
assisted by IF-1 and IF-3 perhaps.
30S subunit likely stays attached to
polycistronic messages and “slides”
to next Shine-Dalgarno sequence.
Lehninger Principles of Biochemistry, 4th ed., Ch 27
Mechanisms ↑translational efficiency
This figure is not drawn to scale…..
RNA polymerase (Mr ~3.9 ×105)
ribosome (Mr ~2.7 × 106)
Lehninger Principles of Biochemistry, 4th ed., Ch 27
Translational efficiency enhanced by
polysomes too (elongation is rate-limiting)
Ribosome recycling
Fig. 27.29
In E. coli, 15,000 ribosomes
synthesizing @ 15 AA/sec →
750 proteins of 300 AA/sec.
One ribosome, one mRNA
model does not account
for the total rate of protein
synthesis per E. coli cell.
As many as 50 ribosomes
bound per mRNA under
certain conditions.
Summary of important differences in
translation machinery in eukaryotes
• Ribosome (polysomes and attached to ER)
– Additional 5.8S rRNA component in large 60S subunit
– mRNA aligned on the small 40S subunit using 5′ cap (no
Shine-Dalgarno sequence or fMet)
– Scanning identifies correct start Met
– No E site, deacylated tRNAs released directly from P site
• Initiation factors (multiple eIFs)
– Many more required (~9-11 vs 3)
– Some bind mRNA, others attach to ribosomal subunits
• Elongation factors (eEFs)
– No differences, all orthologs of prokaryotic EFs
• Termination (only one release factor)
– eRF recognizes all stop codons (UAA, UAG, UGA)
Initiation of translation in eukaryotic
cells:connecting the head and tail
GCCRCCAUGG
1: Multiple initiation factors with distinct biochemical roles (linking, tethering,
recruiting, and scanning)
2: 5′ and 3′ ends of mRNA tied together and tethered to 40S subunit via
eIF/PAB complex. Longer poly (A) tract → more efficient translation
3: Identification of start AUG achieved by “scanning” mechanism involving
eIF4B and eIF4F(complex of 4E, 4A, and 4G). Initiator tRNA is Met-tRNAMet.