Transcript (CH14) Translation (Slides)

Chapter 14:
The Mechanism of
Translation
We can do experiments on rRNAs today
that are far more powerful than anything
we ever attempted with ribosomal
proteins in the past. So today we work
on rRNA; we would be crazy not to.
Peter B. Moore, The Ribosome. Structure,
Function, and Evolution (1990), p. xxi
14.1 Introduction
• Ribosomes are assembled in the
nucleolus of the cell.
• tRNAs are “charged” with their
appropriate amino acids.
• All the players in protein synthesis join
together in the cytoplasm.
14.2 Ribosome structure and
assembly
Ribosomes are the protein synthesis
machinery
• Assemble in the nucleolus of the cell and are
exported to the cytoplasm.
• Decode the genetic code in the mRNA.
• Catalyze the formation of peptide bonds
between amino acids.
“This seems a satisfactory name and
it has a pleasant sound.”
Dick Roberts
(1958, upon proposing the name ribosome)
Structure of ribosomes
• Determined through a combination of Xray crystallography, cryoelectron
microscopy, biochemical, and genetic
data.
• Ribosomes consist of two subunits,
large and small, composed of ribosomal
RNA and many ribosomal proteins.
What is “S”?
•
Ultracentrifugation was developed in the
1920s by Theodor Svedberg.
•
“S” is one Svedberg unit, which equals a
sedimentation coefficient of 10-13 seconds,
during density-gradient centrifugation.
•
S increase with particle mass, but is only a
rough estimate of molecular weight.
Three-dimensional models of ribosomes
• The small subunit includes a head,
base, and platform.
• The large subunit includes the central
protuberance, ridge, and stalk.
•
There are three tRNA binding sites on the
ribosome that bridge the large and small
subunits
– (A) acceptor
– (P) peptidyl
– (E) exit
• The peptidyl transferase center is in the
large subunit.
• Decoding the mRNA occurs on the
small subunit.
The nucleolus
•
Non-membrane-bound subcompartment of
the nucleus.
•
Eukaryotic large and small ribosomal subunits
are assembled within the nucleolus.
•
Site of 45S preribosomal RNA transcription by
RNA polymerase I.
The nucleolus
•
The 45S pre-rRNA is processed to form the
28S rRNA, 18S rRNA, and 5.8S rRNA.
•
5S rRNA is transcribed by RNA pol III.
•
The 28S, 5.8S, and 5S rRNAs are assembled
with ribosomal proteins to form the large
subunit.
•
The 18S rRNA and associated proteins form
the small subunit.
Ribosome biogenesis
•
The earliest precursor particle is the 90S
precursor, associated with partially processed
35S pre-rRNA.
•
Cleavage of the 35S pre-rRNA splits the 90S
precursor into the 40S and 60S pre-ribosomal
subunits.
•
After export into the cytoplasm via the nuclear
pore complexes, the remaining nonribosomal
factors dissociate.
14.3 Aminoacyl-tRNA
synthetases
The overall fidelity of translation is
dependent on the accuracy of two
processes
• Codon-anticodon recognition
• Aminoacyl-tRNA synthesis
Aminoacyl-tRNA charging
• Aminoacyl-tRNA synthetases attach an
amino acid to a tRNA in two enzymatic
steps:
1. The amino acid reacts with ATP to become
adenylylated (addition of AMP) and
pyrophosphate is released.
2. AMP is released and the amino acid is
transferred to the 3′ end of the tRNA.
•
Each aminoacyl-tRNA synthetase is able to
precisely match a particular amino acid with
the tRNA containing the correct
corresponding anticodon.
•
Specific aminoacyl-tRNA synthetases are
denoted by their three-letter amino acid
designation.
– e.g. MetRS for methionyl-tRNA synthetase
Nomenclature
• Methionine tRNA or tRNAMet indicates
the uncharged tRNA specific for
methionine.
• Methionyl-tRNA or Met-tRNA denotes
the tRNA aminoacylated with
methionine.
Experimental example
• Charging of tRNACUA with pyrrolysine.
• A special tRNA called tRNACUA and a
novel archael aminoacyltRNA
synthetase, PylS are required for
incorporation of pyrrolysine.
Proofreading activity of aminoacyltRNA synthetases
• Overall error rate of about one in 10,000
achieved by two means:
– Initial high fidelity selection
– Proofreading
Editing domain of ThrRS
•
ThrS can distinguish between serine and
threonine, despite the similarity in side chains.
•
Water-mediated hydrolysis of the mischarged
tRNA.
•
The correct product, Thr-tRNAThr is not
hydrolyzed due to steric exclusion from the
deep editing pocket.
14.4 Initiation of translation
•
Translation can be divided into three main
stages
– Initiation
– Elongation
– Termination
•
Initiation is the most complex and tightly
controlled step in protein synthesis.
• The process of protein synthesis is
fundamentally the same in bacteria and
eukaryotes.
• The difference lies in the details of some of the
steps and in the components used to accomplish
each step.
• Focus on eukaryotic protein synthesis, with
reference to bacterial protein synthesis for
comparison.
Initiation is subdivided into four steps
1. Ternary complex formation and loading onto
the 40S ribosomal subunit.
2. Loading of the mRNA.
3. Scanning and start codon recognition.
4. Joining of the 40S and 60S subunits to form
the functional 80S ribosomes.
Ternary complex formation and loading
onto the 40S ribosomal subunit
•
•
The ternary complex is composed of:
– Eukaryotic initiation factor 2 (eIF2)
– GTP
– The amino acid-charged initiator tRNA
(Met-tRNA)
The ternary complex binds the 40S ribosomal
subunit, plus other initiation factors, including
eIF4G/E, to form a 43S complex.
Loading the mRNA on the 40S
ribosomal subunit
•
eIF4G and eIF4E, initiation factors with RNA
helicase activity, associate with the 5′ cap of
the mRNA and unwind any secondary and
tertiary structures.
•
Other initiation factors associate with the
poly(A)-binding protein (PABP) bound to the
3′-poly(A) tail.
The closed-loop model of translation
initiation
•
The 5′-cap and 3′-poly(A) tail of the mRNA
join to form a closed loop with eIF4G serving
as the bridge between them.
•
Some cellular RNAs are translated by a 5′cap-independent mechanism in which
ribosomes are directly recruited by an internal
ribosome entry site (IRES).
Scanning and start codon recognition
•
Once the mRNA is loaded, the 43S complex
scans along the message from 5′→3′ looking
for the AUG start codon.
•
ATP-dependent mechanism.
•
AUG is embedded in a Kozak consensus
sequence.
Translation toeprinting assays
•
mRNA is translated using ribosomal
components.
•
The mRNA complex is copied into cDNA by
reverse transcriptase using a complementary
labeled primer.
•
Where the reverse transcriptase meets the
ribosome bound to the mRNA, polymerization
is halted and a “toeprint” cDNA fragment is
generated.
• Toeprinting assays have been used to
characterize the initiation factors
required for AUG recognition in either a
“good” or “bad” sequence context.
Joining of the 40S and 60S ribosomal
subunits to form the functional
80S ribosomes
•
Initiation factors are released from the 43S
complex in a process that requires GTP
hydrolysis.
•
eIF2-GDP is converted to eIF2-GTP through a
nucleotide exchange reaction mediated by
eIF2B.
•
The 60S subunit joins with the 40S subunit to
form the 80S ribosome initiation complex, in a
process that requires a second GTP
hydrolysis step.
•
eIF5-GDP is converted to eIF5-GTP through a
nucleotide exchange reaction mediated by
eIF5B.
Eukaryotic initiation factor 2B and
vanishing white matter
•
Recessively inherited, fatal disease.
•
Progressive loss of movement and speech,
seizures, and coma usually before age 5.
•
Episodes of rapid and major deterioration
following fever or minor head trauma.
•
White matter (myelinated axons) vanishes
and is replaced by cerebrospinal fluid.
•
Mutations in the genes encoding the 5
subunits of guanine nucleotide exchange
factor eIF2B.
•
Why the cells that make myelin are
particularly sensitive to defects in eIF2B
remains unknown.
14.5 Elongation and events in
the ribosome tunnel
• Peptide chain elongation begins with a
peptidyl-tRNA in the ribosomal P site
next to a vacant A site.
• An aminoacyl-tRNA is carried to the A
site as part of a ternary complex with
GTP-bound eEF1A.
• Upon GTP hydrolysis, the aminoacyltRNA enters the A site where decoding
takes place.
• eEF1A-GDP is converted to eEF1AGTP through a nucleotide exchange
reaction mediated by eEF1B.
•
•
The incorporation of selenocysteine at a
specific UGA site depends upon:
– tRNASel
– Elongation factor SelB and SBP2 (in
eukaryotes)
– A selenocysteine insertion sequence
(SECIS)
Pyrrolysine insertion elements (PYLIS) in
archaea signal for pyrrolysine incorporation at
a UAG codon.
Decoding the message
•
tRNA selection involves two steps: initial
selection and proofreading.
•
The two steps are separated by irreversible
hydroylsis of GTP.
•
When the ternary complex contains the
correct tRNA, the initial selection step occurs
more rapidly and GTP hydrolysis releases the
tRNA in the A site.
•
Conformational changes in tRNA are the
basis for induced fit, which is essential for
high-fidelity tRNA selection.
•
Only one incorrect amino acid per 1000 to
10,000 correct amino acids.
Experimental evidence for the importance of
tRNA conformation in decoding
•
Studies of a mutant called the “Hirsch
suppressor,” a tRNATrp variant that has a
single G24A substitution in the D arm.
•
Mutant tRNA recognizes both UGG and UGA
stop codons even though the mutation does
not alter the anticodon.
• The Hirsch suppressor mutant
accelerates the forward rate constants
during decoding independent of codonanticodon pairing.
Peptide bond formation and
translocation
•
Peptidyl transferase activity transfers a
growing polypeptide chain from peptidyl-tRNA
in the P site to an amino acid esterified with
another tRNA in the A site.
•
After the tRNAs and mRNA are translocated
and the next codon is moved to the A site, the
process is repeated.
•
Mediated by eEF2; requires GTP hydrolysis.
Peptidyl transferase activity
•
The site of peptide bond formation is located
at the base of the central protuberance in the
large ribosomal subunit.
•
A central question for many years was
whether the “peptidyl transferase activity” that
catalyzes peptide bond formation is the result
of a protein or RNA enzyme.
Biochemical evidence that 23S rRNA is
a ribozyme
• “Fragment reaction” used by Harry
Noller and colleagues to shown that
purified bacterial 23S rRNA has
“peptidyl transferase activity” in vitro.
Structural evidence that rRNA forms
the active site of the ribosome
•
X-ray crystallographic images at atomic (2.4Å)
resolution of archeon Haloarcula marismortui
large ribsosomal subunits.
•
rRNA forms the catalytic center, decoding
site, A, P and E sites, and the intersubunit
interface.
•
Ribosomal proteins are abundant on the
exterior of the ribosome.
The ribosome is a ribozyme
•
The peptidyl transferase center is located in
domain V of the 23S rRNA.
•
One model proposes that the 107-fold rate of
enhancement of peptide bond formation is
due to substrate positioning by the 23S rRNA
within the active site, rather than chemical
catalysis.
• Universally conserved nucleotides in the
peptidyl transferase center are critical
for the catalysis of peptide release
during termination.
Events in the ribosome tunnel
•
Nascent proteins move through a long
“tunnel” from the site of peptidyl transferase
activity to the peptide exit hole.
•
Proteins emerging from the ribosome tunnel
often associate with other factors.
•
These factors connect proteins to
downstream processes or act as folding
chaperones.
•
Cotranslation translocation pathway from the
ribosome to the endoplasmic reticulum
(ER) lumen.
•
Signal recognition particle (SRP) binds to a
ribosome translating a polypeptide that bears
a signal sequence for targeting to the ER.
•
The SRP and SRP receptor use a cycle of
recruitment and hydrolysis of GTP to control
delivery of the ribosome-mRNA complex to
the ER translocon.
The E. coli trigger factor
• Creates a protected folding space where
nascent polypeptides are shielded from
proteases and aggregation as they
emerge from the peptide exit hole.
14.6 Termination of translation
•
The stop codons are recognized by release
factor eRF1 in association with eRF3.
•
The completed polypeptide is cleaved from
the peptidyl-tRNA.
•
Dissociation of the ribosome from the mRNA,
and dissociation of the 40S and 60S subunits.
•
GTP hydrolysis may trigger the release of
eRF1 and eRF3.
14.7 Translational and posttranslational control
• Additional levels of gene regulation in
eukaryotes.
• A classic example of both levels of
control is the phosphorylation of eIF2.
Phosphorylation of eIF2 blocks
ternary complex formation
•
Hypoxia, viral infection, amino acid starvation,
heat shock, etc. trigger the phosphorylation of
the -subunit of eIF2.
•
Phosphorylation of eIF2 inhibits GDP-GTP
exchange.
•
Reduces the dissociation rate of the
nucleotide exchange factor eIF2B.
•
eIF2 phosphorylation leads to inhibition of
translation by blocking ternary complex
formation.
•
Selective translation of a subset of mRNAs
continues, which allows cells to adapt to
stress conditions.
eIF2 phosphorylation is mediated by
four distinct protein kinases
• Four distinct protein kinases are
activated in response to different stress
conditions:
– Heme-regulated inhibitor kinase (HRI)
– Protein kinase RNA (PKR)
– PKR-like endoplasmic reticulum kinase
(PERK)
– General control non-depressible 2 (GCN2)
Model of the protein kinase RNA (PKR)
activation pathway:
•
•
•
•
•
Viral double-stranded RNA binds to the RNA
binding domains of PKR.
PKR catalytic-domain dimerization.
Autophosphorylation of PKR.
Specific recognition of eIF2.
Phosphorylation of eIF2.
Model of the protein kinase RNA (PKR)
activation pathway
• HRI is activated by low heme levels.
• Activated HRI phosphorylates eIF2.
• Prevents the synthesis of globin in
excess of heme.
Experimental example:
•
Protein synthesis and eIF2 phosphorylation
in reticulocytes from HRI/ knockout mice
•
In iron-deficient HRI/ knockout mice, much
of eIF2 remains unphosphorylated.
•
Synthesis of both  and -globin continues,
resulting in aggregation of globin in red blood
cells, anemia, and accelerated apoptosis in
bone marrow and spleen.