Amino-acyl tRNA synthetases:

One synthetase for each amino acid

a single synthetase may recognize multiple tRNAs
for the same amino acid

Two classes of synthetase.

Different 3-dimensional structures
Differ in which side of the tRNA they recognize
and how they bind ATP
Class I - monomeric, acylates the 2’OH on the terminal ribose
Arg, Cys , Gln, Glu, Ile, Leu, Met, Trp Tyr, Val

Class II - dimeric, acylate the 3’OH on the terminal ribose

Ala, Asn, Asp, Gly, His, Lys, Phe, Ser, Pro, Thr
tRNAs are activated by amino-acyl tRNA synthetases
Two levels of control to ensure that the proper amino acid
is incorporated into protein: 1) Charging of the proper tRNA
2) Matching the
cognate tRNA to the
messenger RNA
Incorporation of amino acids into polypeptide chains I
Incorporation of amino acids into polypeptide chains II
In addition to the APE sites there is an mRNA binding groove
that holds onto the message being translated
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 fMet-tRNAfMet can bind only in the P site &
that no other aa-tRNA 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 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 and delivers an aminoacyl-tRNA
to the A site on the ribosome.
EF-Tu recognizes & binds all aminoacyl-tRNAs
with approximately the same affinity, when each
tRNA is bonded to the correct (cognate) amino acid.
=EF-1 Proper reading of the
anticodon is the second
important quality control
step ensuring accurate
protein synthesis

Elongation factors
Introduce a two-step
“Kinetic proofreading”
A second elongation factor
EF-G or EF-2, drives the
translocation of the ribosome
along the mRNA

Together GTP hydrolysis

by EF-1 and EF-2 help drive
protein synthesis forward
 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.
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, and 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
For images depicting the
the mouth of the tunnel proposed rotational
through which nascent poly- movement, see Fig. 5B in
peptides exit the ribosome. website of A. E. Yonath.
 EF-Tu-GTP*

GDP

EF-Ts (GEF) ribosome (GAP)

EF-Ts
functions as GTP Pi
GEF to EF-Tu-GDP**
reactivate
EF-Tu. *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.
Transpeptidation (peptide bond formation)
involves acid/base catalysis by a universally
conserved adenosine of the 23S rRNA of the large
ribosomal subunit.
The 23S rRNA may be considered a "ribozyme.“
The amino N of the amino acid linked to the 2' or 3'
OH of the terminal adenosine of tRNA in the A site
reacts with the carbonyl C of the amino acid (with
attached nascent polypeptide) linked to the tRNA in
the P site.
 tRNA P site tRNA A site
O O
Adenine O P O CH2 Adenine
O P O CH2 O
O −
O H H
O− H H
H H H H
O OH O OH

O C O C

HC R HC R

NH :NH2

O C
HC R
NH3+

A ring N of the catalytic adenosine may promote the

reaction by extracting a H+ from the attacking amino N.
This H+ is then donated to the hydroxyl of the tRNA in
the P site, as the ester linkage is cleaved.
The nascent
tRNA P site tRNA A site
polypeptide, one O O
residue longer, O P O CH2 Adenine O P O CH2 Adenine
O O
is now linked to O − H H O − H H
the A-site tRNA. H
OH
H
OH
H
O
H
OH
O C
Translocation
HC R
has already NH
partly occurred, O C
because of the HC R
earlier rotation NH
of the 3' end of
the A-site tRNA O C
toward the HC R
P-site, prior to NH3+

catalysis.
Peptide bond formation is catalyzed by the large subunit rRNA
Peptide bond formation is catalyzed by the large subunit rRNA
 tRNA grey,
EF-Tu red,
EF-G blue

The unloaded tRNA in the P site will shift to an exit

(E) site during translocation.
Translocation of the ribosome relative to mRNA
involves the GTP-binding protein EF-G.
 large subunit tRNA

EF-G

mRNA
small subunit 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 an exit site.
Since tRNAs are linked to mRNA by codon-anticodon base
pairing, the mRNA would move 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.
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.
Termination of translation
is triggered by stop codons

Release factor enters

the A site and triggers
hydrolysis the peptidyl-tRNA
bond leading to release of
the protein.
Release of the protein causes
the disassociation of the
ribosome into its constituent
subunits.
Ribosome Assembly

The proteins of each

ribosomal subunit
are organized around
rRNA molecules

16S rRNA
23S rRNA secondary structure