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Molecular Biochemistry II
Protein Synthesis
Copyright © 1999-2008 by Joyce J. Diwan.
All rights reserved.
The focus here will be on structural aspects, and on
protein factors involved in initiation, elongation, &
termination of protein synthesis.
Many of these factors are GTP-binding proteins, &
other proteins that control GDP/GTP exchange or
GTPase activity of these GTP-binding proteins.
Bacterial translation mechanisms will be emphasized.
The more complex process of mammalian translation
and its regulation will be only briefly introduced.
Heterotrimeric G-proteins, and the related family of
small GTP-binding proteins, were introduced in the
notes on cell signals.
 A GTP-binding protein has a different conformation
depending on whether it has bound to it GTP or GDP.
 Usually bound GTP stabilizes the active conformation.
 Hydrolysis of the bound GTP to GDP + Pi converts
the protein to the inactive conformation.
 Reactivation occurs by release of bound GDP in
exchange for GTP.
Small GTP-binding
proteins require
helper proteins, to
• facilitate
GDP/GTP
exchange, or
• promote GTP
hydrolysis.
G protein-GTP (active)
GDP
GEF
GTP
GAP
Pi
G protein-GDP (inactive)
A guanine nucleotide exchange factor (GEF) induces a
conformational change that makes the nucleotide-binding
site of a GTP-binding protein more accessible to the
aqueous intracellular milieu, where [GTP]  [GDP].
Thus a GEF causes a GTP-binding protein to release
GDP & bind GTP (GDP/GTP exchange).
A GTPase
activating
protein (GAP)
causes a GTPbinding protein
to hydrolyze its
bound GTP to
GDP + Pi.
G protein-GTP (active)
GDP
GEF
GAP
GTP
Pi
G protein-GDP (inactive)
The active site for GTP hydrolysis is on the GTP-binding
protein, although a GAP may contribute an essential active
site residue.
GEFs & GAPs may be separately regulated.
Unique GEFs and GAPs interact with different
GTP-binding proteins
Members of the family of small GTP-binding proteins
have diverse functions.
In some cases, the difference in conformation, with
substitution of GDP for GTP allows a GTP-binding
protein to serve as a "switch".
In other cases the conformational change may serve a
mechanical role or alter the ability of the protein to
bind to membranes.
Initiation of protein synthesis in E. coli requires
initiation factors IF-1, IF-2, & IF-3.
 IF-3 binds to the 30S ribosomal subunit, freeing it
from its complex with the 50S subunit.
 IF-1 assists binding of IF-3 to the 30S ribosomal
subunit.
IF-1 also occludes the A site of the small ribosomal
subunit, helping insure that the initiation aa-tRNA fMettRNAfMet can bind only in the P site & that no other aatRNA can bind in the A site during initiation.
 IF-2 is a small GTP-binding protein.
IF-2-GTP binds the initiator fMet-tRNAfMet & helps
it to dock with the small ribosome subunit.
 As mRNA binds, IF-3 helps to correctly position the
complex such that the tRNAfMet interacts via base
pairing with the mRNA initiation codon (AUG).
A region of mRNA upstream of the initiation codon,
the Shine-Dalgarno sequence, base pairs with the
3' end of the 16S rRNA. This positions the 30S
ribosomal subunit in relation to the initiation codon.
 As the large ribosomal subunit joins the complex,
GTP on IF-2 is hydrolyzed, leading to dissociation of
IF-2-GDP and dissociation of IF-1.
A domain of the large ribosomal subunit serves as
GAP (GTPase activating protein) for IF-2.
 Once the two ribosomal subunits come together, the
mRNA is threaded through a curved channel that
wraps around the "neck" region of the small subunit.
Elongation cycle
Ribosome structure
and position of
factors & tRNAs
based on cryo-EM
with 3D image
reconstruction.
Diagram provided
by Dr. J. Frank,
Wadsworth Center,
NYS Dept. of Health.
Partial images on
subsequent slides are
derived from this.
Colors: large ribosome subunit, cyan; small subunit, pale yellow;
EF-Tu, red; EF-G, blue. tRNAs, gray, magenta, green, yellow, brown.
Elongation requires participation of elongation factors
• EF-Tu (also called EF1A)
• EF-Ts (EF1B)
• EF-G (EF2)
EF-Tu & EF-G are small GTP-binding proteins.
The sequence of events follows.
EF-Tu-GTP binds & delivers an
aminoacyl-tRNA to the A site
on the ribosome.
EF-Tu recognizes & binds all
aminoacyl-tRNAs with approx.
the same affinity, when each
tRNA is bonded to the correct
(cognate) amino acid.
tRNAs for different amino acids
have evolved to differ slightly
EF-Tu colored red
in structure, to compensate for
different binding affinities of amino acid side-chains, so the
aminoacyl-tRNAs all have similar affinity for EF-Tu.
The tRNA must have the correct anticodon to interact
with the mRNA codon positioned at the A site to form a
base pair of appropriate geometry.
Universally conserved bases of 16S rRNA interact with
and sense the configuration of the minor groove of the
short stretch of double helix formed from the first 2 base
pairs of the codon/anticodon complex.
A particular ribosomal conformation is stabilized by this
interaction, providing a mechanism for detecting whether
the correct tRNA has bound.
Proofreading in part involves release of the aminoacyltRNA prior to peptide bond formation, if the appropriate
ribosomal conformation is not generated by this
interaction.
EF-Tu-GTP
ribosome (GAP)
Pi
EF-Tu-GDP
The change in ribosomal conformational associated
with formation of a correct codon-anticodon complex
leads to altered positions of active site residues in the
bound EF-Tu, with activation of EF-Tu GTPase
activity.
The ribosome thus functions as GAP for EF-Tu.
When EF-Tu delivers an
aminoacyl-tRNA to the
ribosome, the tRNA initially
has a distorted conformation.
As GTP on EF-Tu is
hydrolyzed to GDP + Pi ,
EF-Tu undergoes a large
conformational change &
dissociates from the complex.
The tRNA conformation
relaxes, & the acceptor stem
is repositioned to promote
peptide bond formation.
This process is called accommodation.
EF-Tu colored red
It includes rotation of the
single-stranded 3' end of the
acceptor stem of the A-site
tRNA around an axis that
bisects the peptidyl transferase
center of the ribosomal large
subunit.
This positions the 3' end with
its attached amino acid in the
active site, near the 3' end of
the P-site tRNA, & adjacent to
the mouth of the tunnel
through which nascent polypeptides exit the ribosome.
PDB 1GIX
acceptor stems
of P-site &
A-site tRNAs
For images depicting the
proposed rotational
movement, see Fig. 5B in
website of A. E. Yonath.
EF-Tu-GTP*
GDP
EF-Ts (GEF)
ribosome (GAP)
GTP
EF-Ts
functions as
GEF to
reactivate
EF-Tu.
Pi
EF-Tu-GDP**
*EF-Tu-GTP (conformation 1) binds &
delivers aa-tRNA to A site on ribosome.
**EF-Tu-GDP (conformation 2)
dissociates from complex.
Interaction with EF-Ts causes EF-Tu to release GDP.
Upon dissociation of EF-Ts, EF-Tu binds GTP, which is
present in the cytosol at higher concentration than GDP.
O
N

O
O
H
O
P
N
P
O
O
N
O
O
P
O
CH2
O
H
NH
N
NH2
O
H
H
OH
H
OH
GDPNP
The difference in conformation of EF-Tu, depending on
whether GDP or GTP occupies its nucleotide binding site,
is apparent in crystal structures to be viewed by Chime.
In 2 of the crystals, GDPNP, a non-hydrolyzable analog
of GTP, is present in the nucleotide-binding site of EF-Tu.
Compare, using Chime, the structures of:
 EF-Tu with bound GDP
 EF-Tu with bound GTP analog GDPNP
 EF-Tu with bound GTP analog & Phe-tRNAPhe
Work in groups of 3, with one of the 3 files assigned to
each student in the group.
Please use colors and displays exactly as specified, so that
images can be compared.
Each student should examine all 3 structures by observing
displays prepared by other group members.
Question: Does substitution of GTP (GDPNP) for GDP,
or binding of aa-tRNA, affect EF-Tu conformation more?
tRNA
P site
tRNA
A site
Transpeptidation
O
O
Adenine
Adenine O P O CH
(peptide bond
O P O CH
O
O
H
H
O
H
H
O
formation) involves
H
H
H
H
O
OH
O
OH
nucleophilic attack
O C
O C
of the amino N of
HC R
HC R
the amino acid
:NH
NH
linked to the 3'OH
of the terminal
O C
HC R
adenosine of the
NH
tRNA in the A site
on the carbonyl C of the amino acid (with attached nascent
polypeptide) in ester linkage to the tRNA in the P site.
2
2


2
3
+
The reaction is promoted by the geometry of the active site
consisting solely of residues of the 23S rRNA of the large
ribosomal subunit. No protein is found at the active site.
tRNA
P site
O
O
O
P
A site
tRNA
O CH2
O
H
O
O
H
H
O
H
OH
O
P
O
O CH2

H
O
C
HC
R
Adenine
O
H
H
O
H
OH
C
HC
R
:NH2
NH
O
Adenine
C
HC
R
NH3+
The 23S rRNA may be considered a "ribozyme."
As part of the reaction a proton (H+) is extracted from the
attacking amino N.
tRNA
P site
O
O
O
P
A site
tRNA
O CH2
O
H
Adenine
O
H
H
OH
H
OH
O
P
O CH2
O
H
O
Adenine
O
H
H
O
H
OH
C
HC
R
NH
O
C
HC
R
NH
O
C
HC
R
NH3+
This H+ is then donated to the hydroxyl of the tRNA in the
P site, as the ester linkage is cleaved.
It had been proposed that a ring N of a highly conserved
adenosine at the active site might act as a catalyst
mediating this H+ transfer.
However, on the basis of recent structural and mutational
evidence it has been concluded that the active site adenine
is essential only as part of the structure of the active site
that positions the substrates correctly.
H+ shuttling is attributed instead to the adjacent ribose 2'
hydroxyl group of the P-site peptidyl-tRNA, along with
ribose hydroxyls of active site rRNA residues & structured
water molecules that collectively form a H-bonded
network at the active site.
For a diagram see Fig 5 of the review by Rodnina et al.
tRNA
P site
O
O
O
P
O
O CH2

H
The nascent
polypeptide, one
residue longer,
is now linked to
the A-site tRNA.
A site
tRNA
Adenine
O
H
H
OH
H
OH
O
P
O
O CH2

H
O
O
H
H
O
H
OH
C
HC
R
NH
O
C
HC
R
NH
O
Adenine
C
HC
R
NH3+
However, translocation has already partly occurred,
because peptide bond formation is associated with
rotation of the single-stranded 3' end of the A-site tRNA
toward the P-site, positioning the aminoacyl moiety for
catalysis.
This rotary movement also positions the nascent
polypeptide to feed into the entrance to the protein exit
tunnel, which is located midway between A & P sites.
tRNA grey,
EF-Tu red,
EF-G blue
The unloaded tRNA in the P site will shift to the E (exit)
site during translocation.
Translocation of the ribosome relative to mRNA involves
the GTP-binding protein EF-G.
The size & shape of EF-G are comparable to that of the
complex of EF-Tu with an aa-tRNA.
Structural studies & molecular dynamics indicate that
EF-G-GTP binding in the vicinity of the A site causes a
ratchet-like motion of the small ribosomal subunit against
the large subunit.
large subunit
tRNA
EF-G
small subunit
mRNA
location
Figure provided by Dr. J. Frank, Wadsworth Center.
The tRNA with attached nascent polypeptide is pushed
from the A site to the P site.
Unloaded tRNA that was in the P site shifts to the E site.
Since tRNAs are linked to mRNA by codon-anticodon base
pairing, the mRNA moves relative to the ribosome.
Additionally, it has been postulated that translocation is
spontaneous after peptide bond formation because:
• the deacylated tRNA in the P site has a higher
affinity for the E site, &
• the peptidyl-tRNA in the A site has a higher affinity
for the P site.
Interaction with the ribosome, which acts as GAP
(GTPase activating protein) for EF-G, causes EF-G to
hydrolyze its bound GTP to GDP + Pi.
EF-G-GDP then dissociates from the ribosome.
A domain of EF-G functions as its own GEF (guanine
nucleotide exchange factor) to regenerate EF-G-GTP.
The continued codon-anticodon base paring of the
tRNA in the E site is postulated to have a role in
preventing potentially serious frame-shift errors, e.g.,
such as would occur if the tRNAs were to able to shift
laterally by one base pair.
Normally the empty tRNA is released from the E site only
after binding of the correct aminoacyl-tRNA at the A site
causes a decreased affinity for tRNA in the E site
Explore with Chime the 30S moiety of a bacterial
ribosome, complexed with a short, genetically engineered
mRNA, and with tRNAPhe in the A, P, & E sites.
Chain termination requires release factors RF-1,
RF-2, & RF-3. RF-3 is a small GTP-binding protein.
 RF-1 & RF-2 recognize & bind to STOP codons.
One or the other binds when a stop codon is reached.
 RF-3-GTP facilitates binding of RF-1 or RF-2 to
the ribosome.
 Once release factors occupy the A site, Peptidyl
Transferase catalyzes transfer of the peptidyl group
to water (hydrolysis).
 Hydrolysis of GTP on RF-3 causes a conformational
change that results in dissociation of release factors.
 A ribosomal recycling factor (RRF) is required, with
EF-G-GTP and IF-3, for release of uncharged tRNA
from the P site, and dissociation of the ribosome from
mRNA with separation of the two ribosomal subunits.
Websites with animations:
Animation of protein elongation
from the laboratory of J. Frank of the Wadsworth Center, based on
Cryo-EM and X-Ray observations of structures of the ribosome,
elongation factors, and tRNA.
Animation of the ribosome in translation
from the laboratory of V. Ramakrishnan of the MRC Laboratory
of Molecular Biology, based on crystal structures of the ribosome
and various protein factors.
Eukaryotic Translation
Translation of mRNA is highly regulated in multicellular eukaryotic organisms, whereas in prokaryotes
regulation occurs mainly at the level of transcription.
There is global regulation of protein synthesis.
 E.g., protein synthesis may be regulated in relation to
the cell cycle or in response to cellular stresses such
as starvation or accumulation of unfolded proteins in
the endoplasmic reticulum.
 Mechanisms include regulation by signal-activated
phosphorylation or dephosphorylation of initiation
and elongation factors.
Translation of particular mRNAs may be inhibited by
small single-stranded microRNA molecules about 20-22
nucleotides long.
MicroRNAs bind via base-pairing to 3' un-translated
regions of mRNA along with a protein complex RISC
(RNA-induced silencing complex), inhibiting translation
and in some cases promoting mRNA degradation.
 Tissue-specific expression of particular genome-encoded
microRNAs is an essential regulatory mechanism
controlling embryonic development.
 Some forms of cancer are associated with altered
expression of microRNAs that regulate synthesis of
proteins relevant to cell cycle progression or apoptosis.
Protein factors that mediate & control translation are
more numerous in eukaryotes than in prokaryotes.
Eukaryotic factors are designated with the prefix "e".
 Some factors are highly conserved across kingdoms.
E.g., the eukaryotic elongation factor eEF1A is
structurally and functionally similar to the prokaryotic
EF-TU (EF1A).
 In contrast, eEF1B, the eukaryotic equivalent of the
GEF EF-Ts, is relatively complex, having multiple
subunits subject to regulatory phosphorylation.
Initiation of protein synthesis is much more complex in
eukaryotes, & requires a large number of protein factors.
Some eukaryotic initiation factors (e.g., eIF3 & eIF4G)
serve as scaffolds, with multiple domains that bind other
proteins during assembly of large initiation complexes.
Usually a pre-initiation complex forms, including:
 several initiation factors
 the small ribosomal subunit
 the loaded initiator tRNA, Met-tRNAiMet.
This then binds to a separate complex that includes:
 mRNA
 initiation factors including ones that interact with
the 5' methylguanosine cap & the 3' poly-A tail,
structures unique to eukaryotic mRNA.
 Within this complex mRNA is thought to circularize
via interactions between factors that associate with the
5' cap & with a poly-A binding protein.
A simplified diagram of the eukaryotic initiation complex once it
has reached the initiation codon is found in the WormBook.
After the initiation complex assembles, it translocates
along the mRNA in a process called scanning, until the
initiation codon is reached.
Scanning is facilitated by eukaryotic initiation factor
eIF4A, which functions as an ATP-dependent helicase
to unwind mRNA secondary structure while releasing
bound proteins.
A short sequence of bases adjacent to the AUG initiation
codon may aid in recognition of the start site.
After the initiation codon is recognized, there is
hydrolysis of GTP and release of initiation factors, as
the large ribosomal subunit joins the complex and
elongation commences.
See Fig. 1 of the article by Hinnebusch (requires TIBS
subscription).
Some eukaryotic mRNAs have what is called an
internal ribosome entry site (IRES), far from the 5'
capped end, at which initiation may occur without the
scanning process.