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Objectives• Know the structure of mRNA, tRNA, and main characteristics of
ribosomes.
• Know and understand the steps involved in translation
initiation, elongation, and termination.
• Know ansd understand how translation is regulated globally or
in an mRNA specific manner.
Reading: Lodish 6th edition,
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Protein Synthesis
The polymerization of amino acids for protein synthesis is carried out by the ribosome, a large
4 MD ribonucleoprotein complex. The formation of the peptide bond in the peptidyl
transferase center of the large ribosomal subunit is catalyzed by rRNA. In this regard, the
ribosome can be viewed as a ribozyme. However, many proteins are required and serve
structural or regulatory functions.
The four main steps in translation are:
1) Initiation
2) Elongation
3) Termination
4) Recycling
Many of the fundamental steps in translation are conserved between eukaryotes and
prokaryotes. This is particularly true for the elongation step of translation. The process of
initiation, however, varies significantly and is the main step at which regulatory mechanisms
control the global rate of translation or the translation of specific messages in the cell.
Although the ribosome is regarded as the protein synthesis machine, it requires translation
factors to engage with the mRNA, to select activated building blocks for polypeptide
synthesis, to terminate translation, and to recycle ribosomal subunits to new
templates.
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Structure of Ribosome Subunits
Prokaryotes:70S Ribosome; large 50S SU contains 23S rRNA, 5S rRNA, and about 34 different proteins;
small 30SU, 16S rRNA and 21 different proteins. MW= 2,500,000
Eukaryotes: 80S, large 60 SU contains 28S rRNA, 5.8S rRNA, 5S rRNA, and 49 proteins; small 40S
SU contains 18S rRNA and 33 proteins. MW= 4,200,000
About two thirds of the mass is RNA and one third is protein. The structure of the RNAs determine the
overall shape of the ribosome.
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Structure of the tRNA
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Structure of glutaminyl-tRNA synthetase with its
tRNA. The enzyme firmly grips the anticodon,
spreading the three bases widely apart for better
recognition. At the other end, the enzyme unpairs
one base at the beginning of the chain, seen
curving upward here, and kinks the long acceptor
end of the chain into a tight hairpin, seen here
curving downward. This places the 2' hydroxyl on
the last nucleotide in the active site, where ATP
and the amino acid (not present in this structure)
are bound. The amino acid is first linked to AMP
forming the adenylated amino acid, which then is
transferred to a hydroxyl group on the sugar at the
3’end of the tRNA (activated ester linkage)
3’ 5’
3’5’
mRNA
anticodon
tRNA
codon
wobble
position
tRNA structure,
anticodon – green,
acceptor end - red
Wobble base pairing
between codon and
anticodon.
In eukaryotes
wobble codon base/
anticodon base:
U/G or I
C/G or I
A/U
G/C
Aminoacyl-tRNA synthetases, tRNA, and Wobble base-pairing
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The amino acid is first linked to AMP forming the adenylylated amino acid, which is then transferred
to a hydroxyl group on the sugar at the 3’end of the tRNA (activated ester linkage).
Linking of Amino Acids to Specific tRNAs by Aminoacyl-tRNA Synthetase
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Structure of the messenger RNA
There are many structural features of the mRNA that play important roles in the process of
translation, especially in the steps of initiation and perhaps recycling. Unlike prokaryotes, the
eukaryotic mRNAs lack the Shine-Dalgarno sequence. Instead, the small ribosomal SU interacts
with the 5’end and according to current models scans along the RNA until the initiation codon is
found in a specific sequence context, referred to as the Kozak sequence. The main structural
features required for or regulating the efficiency of translation initiation are the 5’cap and the poly
(A) tail. These are bound by translation factors that recruit the 40S SU to the 5’end of the mRNA.
Other sequences, often located in the 5’ or 3’ UTRs (green ovals), regulate the efficiency of
translation. Secondary structures, like hairpins, near the cap-site can reduce translation
efficiency and special translation factors with RNA unwinding activities are required to resolve
these structures. IRES, internal ribosome entry sites, are used to recruit the “preinitiation
complex” to the mRNA in a cap-independent manner. These sequences are not well defined and
range in length from 7 nucleotides to more than 100. Secondary structure may play a more
prominent role than primary sequence and may be recognized by special proteins that mediate
cap-independent translation initiation. Some mRNAs contain upstream open reading frames
(uORF), which often inhibit translation initiation.
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The 5’cap and interacting proteins
The 5’ cap is recognized by the initiation factor complex eIF4F. This multimeric complex consists
of eIF4E, the cap binding protein; eIF4G, a large protein which interacts with eIF4E, proteins
bound to the poly (A) tail, and other translation initiation factors (considered a scaffold protein),
and eIF4A, containing ATPase and RNA helicase activity. b, c: cap/eIF4E/eIf4G ternary complex,
eIF4E is yellow, eIF4G fragment is blue; short helix in lower right corner (eIF4E), and structures in
eIF4G shown here are induced by the interaction of the two proteins. The m7GDP is stacked in
between tow tryptophan residues. The methyl group is recognized and interacts with eIF4E, the
affinity for GDB/GTP is much reduced compared to methylated GDP.
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Poly (A) and interacting proteins
The mRNA precursor is processed at the 3’ end by specific endonucleolytic cleavage immediately
after transcription. The 5’ fragment will become the mRNA, the 3’ fragment is degraded. This
cleavage is mediated by the Cleavage and Polyadenylation Specificity Factor (CPSF), which
recognizes the polyadenylation signal AAUAAA. CPSF stimulates poly (A) polymerase to add As
to the end. PABPN1 (nuclear poly (A) binding protein) also assists in tethering the polymerase to
the RNA. In the presence of CPSF and PABPN, the polymerase can add up to 250 As to the end of
the message.
PABPN is removed from the mRNA in the nucleus and replaced by the cytoplasmic PABPC upon
which the mRNA is exported to the cytoplasm. PABPC is 70 kDa and contains 4 RNA recognition
motifs, the first two of which are important and sufficient for RNA binding. 12 nucleotides are
required for high affinity binding, but it covers about 25 nucleotides.
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Cap, poly (A), and pseudo-circularization of mRNA
The interaction between PABPC and eIF4G leads to the circularization of the mRNA.
Although the cap is sufficient to recruit translation factors and the 40S ribosomal SU, the
presence of the poly (A) tail stimulates translation initiation significantly. The requirement
for both cap and poly (A) tail ensures that only intact RNAs are translated. In addition,
circularization may facilitate translation re-initiation. The interactions between the cap- and
poly(A) complexes mutually stabilize each other. PABPC also interacts with the ribosome
release factor eRF3, a GTPase involved in translation termination and polypeptide release. A
current model proposes that the simultaneous interaction of PABPC with eIF4G and eRF3
results in close neighborhood of termination codon and the cap-complex, with the 3’UTR
looped out.
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Translation Initiation and Initiation Factors (eIFs)
Initiation can be divided into four
major steps:
1) Formation of a 43S
preinitiation complex
consisting of the 40S SU,
initiation factors, and Met-
tRNAi.
2) Recruitment of the 43S
complex to the capped 5’end
of the mRNA.
3) Scanning of the 5’ UTR and
start codon recognition.
4) Joining of the large 60S SU to
assemble the translation
competent
80S ribosome.
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Translation Elongation and Termination
Translation elongation is extremely conserved between prokaryotes, eukaryotes, and archae
bacteria. Aminoacyl t-RNAs are carried to the A (Acceptor) site as part of a ternary complex with
eEF1A and GTP. Base pairing between codon and anticodon, conformational changes, and GTP
hydrolysis ensure that only correct tRNAs are selected. Base pairing induces three bases from
the rRNA of the small SU to swing out and contact resulting mRNA/tRNA duplex, this activates the
GTPase activity. The peptidyl transferase center (P-site) catalysis formation of the peptide bond.
The following translocation is mediated by eEF2 and GTP hydrolysis. eEF1B is a multi factor
complex, which exchanges the GTP in eEF1A. No exchange factor has been described for eEF2.
eEF3 is unique to fungi and facilitates the release of the de-acylated tRNA from the E-site (Exit
site) and also stimulates the binding of eEF1A and tRNA to the A-site. The release from the E-site
requires ATP hydrolysis. Although, a similar factor has not been found in higher eukaryotes, the
exit from the E-site also requires ATP hydrolysis in these organism suggesting the presence of a
comparable activity to eEF3.
Translation termination is the consequence of stop codon recognition, for which no tRNA is
available. Instead, a class I release factor, eRF1, binds/decodes any of the three stop codons
(UAA, UAG, UGA). The recognition of the stop codon by eRF1 also involves the +4 position. Class
II release factor eRF3, a GTPase, stimulates the function of eRF1 as well as the hydrolysis of the
ester bond linking the polypeptide chain to the P-site tRNA. The hydrolysis requires water
molecules to enter the peptidyl transferase center, whereas elongating ribosomes must keep
water out during amid bond formation. eRF1 is thought to provide a channel for water molecules
to enter the transferase center.
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Model of the prokaryotic ribosome
obtained by X-ray crystallography.
Shown are the small (30S) and large (50S)
subunits as well as the positions of the
acceptor (A), peptidyltransferase (P),
and exit sites. Protein components are
shown in darker color.
Structure of the Ribosome
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Translation Elongation Continued
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Translation Elongation
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Translation Elongation Continued
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Translation Elongation Continued
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Translation Elongation Continued
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Translation Elongation Continued
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The Peptidyl Transferase Reaction
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Peptidyl Transferase Center
(a) Large ribosomal subunit from
H. Marismortui with three intact
tRNAs in the E-, P, and A, site.
rRNA is white, ribosomal proteins
are yellow. (b) active site area
showing the peptidyl product (green)
bound to the A-loop (orange), and the
deacylated product (violet) in the
P-loop (dark blue). The N3 of A2486
is in close proximity to the 3’OH of
the CCA, and the base of U2620 has
moved close to the new peptide
bond and the 3’OH of A76.
Moore and Steitz, Annu. Rev. Biochem., 2003
Steps in the peptidyl transferase
reaction pathway. (a) a-amino group of
A-site substrate is positioned for pro-
R attack on carbonyl group of the ester
bond of the P-site substrate (green).
(b) Model of tetrahedral intermediate
with oxyanion pointing away from
A2486. (c) Structure of products of
peptidyl transferase reaction bound to
the peptidyl transferase center.
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Translation Termination and Release Factors
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Global Control of Translation
Global control of protein synthesis in response to various stress signals is generally achieved
by phosphorylation of translation initiation factors or proteins that regulate them. eIF2 consists
of the three subunits a, b, and g. The g-subunit binds GTP. A specific serine residue in the a-
subunit at position 51 is subject to phosphorylation. The phosphorylation of eF2 blocks the GTP
exchange reaction by reducing the dissociation rate of eIF2 from eIF2B, the GTP exchange
factor. Several stimuli lead to the phosphorylation of eIF2 including, haem depletion (by haem
regulated inhibitor), amino acid starvation (in yeast by GCN2), viral infection (by PKR, protein
kinase activated by double stranded RNA), and ER stress (by PERK).
The interaction of eIF4E with eIF4G is regulated by 4E-binding proteins (4E-BPs), which
competitively interact with eIF4E. Phosphorylation of 4E-BPs prevents association with eIF4E
and thus allows translation initiation. Extracellular signals, like insulin, triggers a cascade
leading to phosphorylation of 4E-BPs. The mammalian target of rapamycin (mTOR) is a critical
kinase that senses extracellular stimuly, amino acid availability, and the oxygen and energy
status to activate translation. It phosphorylates not only the 4E-BPs but also eIF4G and the S6
kinase, which phosphorylates eIF4B and enhances its interaction with eIF3. The MAPK pathway
also leads to phosphorylation of eIFs, e.g. eIF4B.
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Global Control of Translation
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RNA-dependent protein kinase PKR mediated phosphorylation of eIF2a
and shut-down of protein synthesis
Viral infection often leads to the accumulation
of double-stranded RNA byproducts. These
are recognized by PKR, which phosphorylates
eIF2a . Phosphorylated eIF2a forms high-
affinity sequestering complexes with its
nucleotide exchange factor eIF2B, which leads
to shut-down of protein synthesis. PKR
dimerization and autophosphorylation is
required for its catalytic activity.
A) PKR/eIF2a complex.
B) PKR dimer interphase
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Viral pseudosubstrate inhibitors of PKR prevent shut-down
of protein synthesis
Most viruses have devised mechanisms to
prevent eIF2a phosphorylation by competitively
interacting with PKR. For example, Vaccinia
virus encodes the protein K3L, which mimics
the 3-dimensional structure of eIF2a and
competitively blocks eIF2a phosphorylation.
A. Model of K3L/PKR inhibitor complex based
on the binding mode of eIF2a. Regions of
structural similarities are colored grey.
B. Activation segment of PKR and interactions
with eIF2a.
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mRNA Specific Regulation of Translation This type of regulation is carried out by proteins that bind specific sequences or structures in
mRNA, mostly in the 5’ or 3’UTR. These regulatory proteins generally repress translation. Iron
regulatory proteins (IRP) regulate translation of ferritin heavy- and light-chains, which encode
for the iron storage protein. In iron deficient cells, IRPs bind to the iron-responsive element (IRE), a
stem loop motif in the ferritin mRNAs, which blocks recruitment of the 43S complex (steric
hindrance). Other modes of regulation involve those that are mediated by proteins that interact with
the 3’ UTR and recruit proteins that interfere with eIF4 activity. These proteins can be regarded as
mRNA specific 4E-BPs as they all interact with eIF4E. Examples are Maskin, recruited by CPEB
(binds to uridine rich Sequence in maternal RNAs of oocyte), Cup, recruited by Bruno (binds to the
3’UTR of nanos in anterior parts of the developing drosophila embryo), and Bicoid, which directly
binds to an element in the Caudal 3’UTR in the anterior pole.
Sex-lethal (SXL) binds to poly (U) sequences in both 5’ and 3’ UTRs of msl-2 mRNA, which encodes
a component of the X-chromosome dosage compensation complex. SXL recruits a co repressor
(CR), which prevents the scanning of cap recruited 40S SU.
Translation of the ceruloplasmin or VEGF mRNAs is repressed by interferon-g. Repression is
mediated by the heteromeric GAIT (IFN-g activated inhibitor of translation) complex. GAIT is
composed of the 60S subunit protein L13a, glutamyl-, prolyl-tRNA synthetase, NS-1 associated
protein 1 (NSAP1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). IFN-g phosphorylates
L13a, which thendissociates from the ribosomal 60S subunit and forms the GAIT complex.
Phosphorylated L13a interacts with eIF4G and blocks its interaction with eIF3, thus inhibiting
recruitment of the 43S PIC.
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mRNA Specific Regulation of Translation
Sonenberg, N, and Hinnebusch, A. G., Cell, 2009
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Translation Control by Micro RNAs
Micro RNAs (miRNAs) are synthesized as primary transcripts in the nucleus and are processed
by Drosha, an RNase-III member. The 70 nucleotide pre-miRNA has a stem loop structure and is
exported to the cytoplasm. Here it is further processed by dicer into 22 nucleotide long miRNAs.
Dicer also generates siRNA, which degrade mRNAs due to perfect complementarity with the
target RNA and recruitment of the RISC complex (RNA induced silencing complex). A component
of the RISC complex is Argonaut, which is also part of the miRNA containing ribonucleoprotein
complex that repress translation. The main difference between miRNAs and siRNAs is the fact
that miRNAs are not perfectly complementary to the mRNA. If they are generated to be perfectly
complementary to the mRNA they behave like siRNAs and degrade the message. In contrast,
miRNAs bind to the 3’UTRs of target mRNAs and repress translation without degrading the RNA.
The mechanism by which translation is inhibited is unknown but must involve an elongation or
termination step as miRNAs were found to interact with polysomes. Initially miRNAs were
identified in Caenorhabditis elegans where they regulate expression of genes that are crucial for
developmental timing.
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Interaction of signal recognition particle (SRP) with ribosome
The existence of signal sequence binding protein
for targeting to the endoplasmatic reticulum (ER) was
first proposed in the early 70’s. The binding
factor was subsequently identified as an 11S
ribonucleoprotein named Signal Recognition
Particle (SRP). The SRP has three activities. It binds
to the signal sequence of newly translated proteins,
it pauses translation elongation, and it promotes
translocation through the ER membrane by docking
to the SRP receptor at the ER. SRP has two domains.
The S domain consists of one half of the 7S RNA and
several proteins. SRP 54 protein is involved in
recognizing the signal sequence and interacts with
the SRP receptor in a GTP-dependent manner.
The signal sequence is recognized near the peptide
exit site of the large ribosomal SU. The Alu domain
consists of 5’ and 3’parts of the 7S RNA and several
proteins. It mediates elongation arrest to allow docking
of the ribosome to the SRP receptor.
Cryo-EM map of SRP bound to the RNC
(ribosome nascent chain complex). 40S SU
in yellow, 60S SU in blue, P-site tRNA in green,
SRP in red. C1-C6: assigned positions of RNC-SRP
Contacts; h1 and h2, hinges of the SRP 7S RNA; ST,
Stalk, SB, stalk base.Halic et al., Nature, 2004
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Halic et al., Nature, 2004
Signal Sequence Dependent SRP Ribosome Interactions
SRP 54 has three domains, the N-terminus (N), the GTPase domain (G) and a methionine rich C-
terminal domain that interacts with the signal sequence. The binding induces a conformational change,
a kink in the 7S RNA possibly mediated by SARP 68/72, that allows interaction of the Alu domain with
the elongation factor binding site in the intersubunit space, where it causes arrest by competing with
elongation factors (EFS, elongation factor binding site).
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Binding of Tetracycline to the 30S Prokaryotic Ribosomal SU
Several antibiotics, like tetracycline,
streptomycin, and hygromycin B all
target functionally important regions
in the 30S SU, mainly rRNA rich
regions associated with tRNA
interaction or movement through the
ribosome. Shown here is the
binding of tetracycline (Tet) to the
30S SU.
(a) Overview of primary and
secondary Tet binding sites (red).
The primary interaction of Tet is with
helix h34 of the 16S rRNA. The binding
site overlaps with the binding site
of the A-site tRNA and inhibits AA-tRNA
during the accommodation step, not the
initial binding of the ternary complex.
(b) Close-up view of the primary
binding site, with the position of the
A-site tRNA (red), mRNA (yellow),
and several helices of the rRNA.
(c) The secondary binding site does not
appear to play a role in Tet mediated
inhibition
Wilson and Nierhaus, 2003
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Antibiotics targeting the large subunit
Crystal structures with antibiotics soaked into the crystal
of the large prokaryotic ribosomal SU have been solved with
14-membered macrolides (e.g. erythromycin) 16-membered
macrolides (e.g. carbomycin), 15-membered macrolides
(azithromycin) and nonmacrolide (e.g. blasticidin and
chloramphenicol) antibiotics. All of these antibiotics
bind in the proximal part of the polypeptide exit tunnel
adjacent to the PTC.
Moore and Steitz, 2003
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Literature: Translation
1. Preiss, T., and M. W. Hentze (2003) Starting the protein synthesis
machine: eukaryotic translation initiation. BioEssays 25:1201-1211.
2. Von der Haar, T., Gross, J.D., Wagner, G., and J.E.G. McCarthy
(2004) The mRNA cap-binding protein eIF4E in post-transcriptional
gene expression. Nat. Struct. & Mol. Biol. 11:503-511.
3. Kuhn, U., and E. Wahle (2004) Structure and function of poly (A)
binding proteins. Biochem. Biophys. Acta 1678:67-84.
4. Gebauer, F., and M. W. Hentze (2004) Molecular Mechanisms of
translational control. Nat Review Mol. Cell Biol. 5:827-835.
5. Moore, P.B., and T. A. Steitz (2003) The structural basis of large ribosomal subunit
function. Ann. Rev. Biochem. 72:813-850.
6. Wilson, D. N., and K. H. Nierhaus (2003) The ribosome through the
looking glass. Ang. Chem. Intl. Ed. 42:3464-3486.
7. Halic, et al. (2004) Structure of the signal recognition particle interacting with the
elongation-arrested ribosome. Nature 427:808-814.
8. Hinnebusch, A.G. (2006) eIF3: a versatile scaffold for translation initiation
complexes. Trends in Biochm. Sci.
9. Dar, A.C., et al. (2005) Higher-order substrate recognition of eIF2a by the RNA-
dependent protein kinase PKR. Cell 122:887-900.
10. Chang, Y-F, J. S. Imam, and M. F. Wilkinson (2007) The nonsense-mediated decay
surveillance pathway. Ann. Rev. Biochem. 76:51-74.
11. Sonenberg, N., and A. G. Hinnebusch (2009) Regulation of Translation initiation in
Eukaryotes: Mechanisms and Biological Targets. Cell 136:731-745.