Amino acid activation. The two-step process in which an amino acid (with its side chain denoted by R) is
activated for protein synthesis by an aminoacyl-tRNA synthetase enzyme is shown. As indicated, the energy of
ATP hydrolysis is used to attach each amino acid to its tRNA molecule in a high-energy linkage. The amino acid is
first activated through the linkage of its carboxyl group directly to an AMP moiety, forming an adenylated amino
acid; the linkage of the AMP, normally an unfavorable reaction, is driven by the hydrolysis of the ATP molecule that
donates the AMP. Without leaving the synthetase enzyme, the AMP-linked carboxyl group on the amino acid is
then transferred to a hydroxyl group on the sugar at the 3′ end of the tRNA molecule. This transfer joins the amino
acid by an activated ester linkage to the tRNA and forms the final aminoacyl-tRNA molecule.
Aminoacyl-tRNA. Amino acids are coupled to tRNAs through ester linkages to
either the 2′- or the 3′-hydroxyl group of the 3′-adenosine residue. A linkage to
the 3′-hydroxyl group is shown.
Classes of Aminoacyl-tRNA Synthetases.
Class I and class II synthetases
recognize different faces of the tRNA molecule. The CCA arm of tRNA adopts different
conformations in complexes with the two classes of synthetase.
Transfer RNA-Binding Sites.
(A) Three tRNA-binding sites are present on the 70S
ribosome. They are called the A (for aminoacyl), P (for peptidyl), and E (for exit) sites.
Each tRNA molecule contacts both the 30S and the 50S subunit. (B) The tRNA
molecules in sites A and P are base paired with mRNA.
Signals for translation initiation Initiation sites in prokaryotic mRNAs are
characterized by a Shine-Delgarno sequence that precedes the AUG initiation
codon. Base pairing between the Shine-Delgarno sequence and a
complementary sequence near the 3´ terminus of 16S rRNA aligns the mRNA on
the ribosome. In contrast, eukaryotic mRNAs are bound to the 40S ribosomal
subunit by their 5´ 7-methylguanosine caps. The ribosome then scans along the
mRNA until it encounters an AUG initiation codon.
Elongation stage of translation The
ribosome has three tRNA-binding sites,
designated P (peptidyl), A (aminoacyl), and E
(exit). The initiating N-formylmethionyl tRNA is
positioned in the P site, leaving an empty A
site. The second aminoacyl tRNA (e.g., alanyl
tRNA) is then brought to the A site by EF-Tu
(complexed with GTP). Following GTP
hydrolysis, EF-Tu (complexed with GDP)
leaves the ribosome, with alanyl tRNA inserted
into the A site. A peptide bond is then formed,
resulting in the transfer of methionine to the
aminoacyl tRNA at the A site. The ribosome
then moves three nucleotides along the
mRNA. This movement translocates the
peptidyl (Met-Ala) tRNA to the P site and the
uncharged tRNA to the E site, leaving an
empty A site ready for addition of the next
amino acid. Translocation is mediated by EFG, coupled to GTP hydrolysis. The process,
illustrated here for prokaryotic cells, is very
similar in eukaryotes
Structure of Elongation Factor Tu.
The structure of a complex between elongation
factor Tu (EF-Tu) and an aminoacyl-tRNA. The amino-terminal domain of EF-Tu is a Ploop NTPase domain similar to those in other G proteins
An aminoacyl tRNA molecule bound to EF-Tu. The three domains of the EF-Tu
protein are colored differently, to match Figure 3-74. This is a bacterial protein;
however, a very similar protein exists in eukaryotes, where it is called EF-1.
Translating an mRNA molecule. Each amino acid
added to the growing end of a polypeptide chain is
selected by complementary base-pairing between the
anticodon on its attached tRNA molecule and the next
codon on the mRNA chain. Because only one of the
many types of tRNA molecules in a cell can base-pair
with each codon, the codon determines the specific
amino acid to be added to the growing polypeptide chain.
The three-step cycle shown is repeated over and over
during the synthesis of a protein. An aminoacyl-tRNA
molecule binds to a vacant A-site on the ribosome in step
1, a new peptide bond is formed in step 2, and the mRNA
moves a distance of three nucleotides through the smallsubunit chain in step 3, ejecting the spent tRNA molecule
and “resetting” the ribosome so that the next aminoacyltRNA molecule can bind. Although the figure shows a
large movement of the small ribosome subunit relative to
the large subunit, the conformational changes that
actually take place in the ribosome during translation are
more subtle. It is likely that they involve a series of small
rearrangements within each subunit as well as several
small shifts between the two subunits. As indicated, the
mRNA is translated in the 5′-to-3′ direction, and the Nterminal end of a protein is made first, with each cycle
adding one amino acid to the C-terminus of the
polypeptide chain. The position at which the growing
peptide chain is attached to a tRNA does not change
during the elongation cycle: it is always linked to the
tRNA present in the P site of the large subunit.
A possible reaction mechanism for the peptidyl transferase activity present in the large ribosomal
subunit. The overall reaction is catalyzed by an active site in the 23S rRNA. In the first step of the proposed
mechanism, the N3 of the active-site adenine abstracts a proton from the amino acid attached to the tRNA at
the ribosome's A-site, allowing its amino nitrogen to attack the carboxyl group at the end of the growing peptide
chain. In the next step this protonated adenine donates its hydrogen to the oxygen linked to the peptidyl-tRNA,
causing this tRNA's release from the peptide chain. This leaves a polypeptide chain that is one amino acid
longer than the starting reactants. The entire reaction cycle would then repeat with the next aminoacyl tRNA that
enters the A-site
Peptide-Bond Formation. The amino group of the aminoacyl-tRNA attacks the carbonyl
group of the ester linkage of the peptidyl-tRNA to form a tetrahedral intermediate. This
intermediate collapses to form the peptide bond and release the deacylated tRNA.
A Role for Formylation. With a free terminal amino group, dipeptidyl-tRNA can cyclize
to cleave itself from tRNA. Formylation of the amino terminus blocks this reaction.
The large conformational change in EF-Tu caused by GTP hydrolysis. (A) The three-dimensional structure of
EF-Tu with GTP bound. The domain at the top is homologous to the Ras protein, and its red α helix is the switch
helix, which moves after GTP hydrolysis, as shown in Figure 3-71. (B) The change in the conformation of the
switch helix in domain 1 causes domains 2 and 3 to rotate as a single unit by about 90° toward the viewer, which
releases the tRNA that was shown bound to this structure in Figure 3-73.
Regeneration of EF-Tu/GTP EF-Tu complexed to GTP escorts the aminoacyl
tRNA to the ribosome. The bound GTP is hydrolyzed as the correct tRNA is
inserted, so EF-Tu complexed to GDP is released. The EF-Tu/GDP complex is
inactive and unable to bind another tRNA. In order for translation to continue, the
active EF-Tu/GTP complex must be regenerated by another factor, EF-Ts, which
stimulates the exchange of the bound GDP for free GTP.
The final phase of protein
synthesis. The binding of a
release factor to an A-site bearing
a stop codon terminates
translation. The completed
polypeptide is released and, after
the action of a ribosome recycling
factor (not shown), the ribosome
dissociates into its two separate
subunits.
comparison of the structures of procaryotic and eucaryotic ribosomes. Ribosomal components are
commonly designated by their “S values,” which refer to their rate of sedimentation in an ultracentrifuge.
Despite the differences in the number and size of their rRNA and protein components, both procaryotic
and eucaryotic ribosomes have nearly the same structure and they function similarly. Although the 18S
and 28S rRNAs of the eucaryotic ribosome contain many extra nucleotides not present in their bacterial
counterparts, these nucleotides are present as multiple insertions that form extra domains and leave the
basic structure of each rRNA largely unchanged.
ribosome at work. (A) The diagram
shows how a ribosome moves along an
mRNA molecule, capturing tRNA
molecules that match the codons in the
mRNA and using them to join amino acids
into a protein chain. The mRNA specifies
the sequence of amino acids. (B) The
three-dimensional structure of a bacterial
ribosome (pale green and blue), moving
along an mRNA molecule (orange beads),
with three tRNA molecules (yellow, green,
and pink) at different stages in their
process of capture and release. The
ribosome is a giant assembly of more
than 50 individual protein and RNA
molecules
Initiation of translation in
eukaryotic cells Initiation factors
eIF-3, eIF-1, and eIF-1A bind to the
40S ribosomal subunit. The initiator
methionyl tRNA is brought to the
ribosome by eIF-2 (complexed to
GTP), and the mRNA by eIF-4E
(which binds to the 5´ cap), eIF-4G
(which binds to both eIF-4E at the 5'
cap and PABP at the 3' poly-A tail),
eIF-4A, and eIF-4B. The ribosome
then scans down the mRNA to
identify the first AUG initiation codon.
Scanning requires energy and is
accompanied by ATP hydrolysis.
When the initiating AUG is identified,
eIF-5 triggers the hydrolysis of GTP
bound to eIF-2, followed by the
release of eIF-2 (complexed to GDP)
and other initiation factors. The 60S
ribosomal subunit then joins the 40S
complex.
Termination of translation A
termination codon (e.g., UAA) at
the A site is recognized by a
release factor rather than by a
tRNA. The result is the release of
the completed polypeptide chain,
followed by the dissociation of
tRNA and mRNA from the
ribosome.
Regulation of translation by
phosphorylation of eIF-2 Translation in
reticulocytes (which is devoted to
synthesis of hemoglobin) is controlled
by the supply of heme, which regulates
the activity of eIF-2. The active form of
eIF-2 (complexed with GTP) escorts
initiator methionyl tRNA to the ribosome
(see Figure 7.10). The eIF-2 is then
released from the ribosome in an
inactive GDP-bound form, which must
be reactivated by exchange of GTP for
the bound GDP. If adequate heme is
available, this exchange occurs and
translation is able to proceed. If heme
supplies are inadequate, however, a
protein kinase that phosphorylates eIF-2
is activated. Phosphorylation of eIF-2
blocks the exchange of GTP for GDP, so
eIF-2/GTP cannot be regenerated and
translation is inhibited.
A possible reaction mechanism for the peptidyl transferase activity present in the large ribosomal subunit.
The overall reaction is catalyzed by an active site in the 23S rRNA. In the first step of the proposed mechanism,
the N3 of the active-site adenine abstracts a proton from the amino acid attached to the tRNA at the ribosome's Asite, allowing its amino nitrogen to attack the carboxyl group at the end of the growing peptide chain. In the next
step this protonated adenine donates its hydrogen to the oxygen linked to the peptidyl-tRNA, causing this tRNA's
release from the peptide chain. This leaves a polypeptide chain that is one amino acid longer than the starting
reactants. The entire reaction cycle would then repeat with the next aminoacyl tRNA that enters the A-site.
The structure of a human translation release factor (eRF1) and its resemblance
to a tRNA molecule. The protein is on the left and the tRNA on the right.
Structure of a Release Factor.
The structure of a eukaryotic release factor reveals a tRNA-like fold. The
acceptor-stem mimic includes the sequence Gly-Gly-Gln at its tip. This region appears to bind a water
molecule, which may be brought into the peptidyl transferase center. There it can participate in the cleavage of
the peptidyl-tRNA ester bond, with the aid of the glutamine residue and the ribosomal catalytic apparatus.
The initiation phase of protein synthesis in eucaryotes. Only three of the many
translation initiation factors required for this process are shown. Efficient translation
initiation also requires the poly-A tail of the mRNA bound by poly-A-binding proteins
which, in turn, interact with eIF4G. In this way, the translation apparatus ascertains that
both ends of the mRNA are intact before initiating (see Figure 6-40). Although only one
GTP hydrolysis event is shown in the figure, a second is known to occur just before the
large and small ribosomal subunits join.
Ribosome assembly Ribosomal proteins are imported to the nucleolus from the
cytoplasm and begin to assemble on pre-rRNA prior to its cleavage. As the prerRNA is processed, additional ribosomal proteins and the 5S rRNA (which is
synthesized elsewhere in the nucleus) assemble to form preribosomal particles.
The final steps of maturation follow the export of preribosomal particles to the
cytoplasm, yielding the 40S and 60S ribosomal subunit