Translation

Translation
• The act of converting information written in one language to other.
• In contrast to copying information in the same language (transcribe).
• In this case, information has to be translated from the linear sequence of the 4-letter
alphabet of the polynucleotide chain into the 20-amino-acid language of the poly-
peptide chain.
• Among the most energetically costly events for the cell.
• In rapidly growing bacterial cells, up to 80% of the cell’s energy and 50% of the cell’s dry
weight are dedicated to protein synthesis.
 Challenges
1. Recognition of 20 distinct amino acids by the enzyme. Did DNA Pol do the same?
2. Reading 3 nucleotides on the template at the same time.
3. Absence of base pair complementarity.
4. Where to attach the enzyme on mRNA?
5. Where to start reading the mRNA for codon?
6. Match speed with Transcription.
7. How many copies of protein to make?
 Messenger RNA & The Genetic Code
• Is the template for translation.
• Contains groups of three-nucleotide-long units, called codons, that code for the 20
different amino acids.
• Order of these codons specify the order of amino acids in the polypeptide chains.
• With 4 possible nucleotides at each position, the total number of permutations of these
triplets is 64 (4 x 4 x 4): well in excess of the number of amino acids.
• Which of these triplet codons are responsible for specifying which amino acids, and what
are the rules that govern their use?
• A striking features of the code is that 61 of the 64 possible triplets specify an amino acid.
• ⇒ Degeneracy
• Codons specifying the same amino acid are synonyms.
TA B L E 16-1 The Genetic Code
second position
U C A G

UUU UCU UAU UGU U

Phe Tyr Cys
UUC UCC UAC UGC C
U Ser
UUA UCA UAA* stop UGA* stop A
Leu
UUG UCG UAG* stop UGG Trp G

CUU CCU CAU CGU U

His
CUC CCC CAC CGC C

third position (3' end)

first position (5' end)
C Leu Pro Arg
CUA CCA CAA CGA A
Gln
CUG CCG CAG CGG G

AUU ACU AAU AGU U

Asn Ser
AUC Ile ACC AAC AGC C
A Thr
AUA ACA AAA AGA A
Lys Arg
AUG† Met ACG AAG AGG G

GUU GCU GAU GGU U

Asp
GUC GCC GAC GGC C
G Val Ala Gly
GUA GCA GAA GGA A
Glu
GUG GCG GAG GGG G

* Chain-terminating or “nonsense” codons.

† Also used in bacteria to specify the initiator formyl-Met-tRNAfMet.
 Messenger RNA

• The protein-coding region(s) of each mRNA is composed of a contiguous, non-

overlapping string of codons called an open reading frame (ORF).
• Each ORF specifies a single protein and starts and ends at internal sites within the mRNA
(the ends of an ORF are distinct from the ends of the mRNA).
• mRNAs contain at least one ORF.
• Eukaryotic mRNAs contain single ORF. Prokaryotic mRNAs may contain two or more
ORFs (can encode multiple polypeptide chains).
• mRNAs containing multiple ORFs are known as polycistronic mRNAs, and those encoding
a single ORF are known as monocistronic mRNAs.
 Messenger RNA: Start and Stop Codons
• Translation starts at the 5’ end of the ORF and proceeds one codon at a time to the 3’
end.
• The first and last codons of an ORF are known as the start and stop codons.
• In Bacteria, the start codon is usually 5’-AUG-3’ (sometimes 5’-GUG-3’ and 5’-UUG-3’).
• In Eukaryotes, always 5’-AUG-3’.
• The start codon has two important functions:
1. It specifies the first amino acid to be incorporated into the growing polypeptide chain.
2. It defines the reading frame for all subsequent codons (out of 3 possible reading
Translation 511 frames).

5' 3'
A U G A A A G C A A U U U U C G U A C U G A A A G G U U G G U G G C G C A C U U C CU G A
M K A I F V L K G W W R T S stop

5' 3'
A U G A A A G C A A U U U U C G U A C U G A A A G G U U G G U G G C G C A C U U C C U G A
stop K Q F S Y stop K V G G A L P

5' 3'
A U G A A A G C A A U U U U C G U A C U G A A A G G U U G G U G G C G C A C U U C C U G A
E S N F R T E R L V A H F L

FIGURE 15-1 Three possible reading frames of the Escherichia coli trp leader sequence.
Start codons are shaded in green, and stop codons are shaded in red. The amino acid sequence
encoded by each reading frame is indicated in the single-letter code below each codon.
 MESSENGER RNA: Start and Stop Codons
• The stop codons are 5’-UAG-3’, 5’-UGA-3’, and 5’-UAA-3’.
• Define the end of the ORF and signal termination of polypeptide synthesis.
Translation 511

5' 3'
A U G A A A G C A A U U U U C G U A C U G A A A G G U U G G U G G C G C A C U U C CU G A
M K A I F V L K G W W R T S stop

5' 3'
A U G A A A G C A A U U U U C G U A C U G A A A G G U U G G U G G C G C A C U U C C U G A
stop K Q F S Y stop K V G G A L P

5' 3'
A U G A A A G C A A U U U U C G U A C U G A A A G G U U G G U G G C G C A C U U C C U G A
E S N F R T E R L V A H F L

FIGURE 15-1 Three possible reading frames of the Escherichia coli trp leader sequence.
Start codons are shaded in green, and stop codons are shaded in red. The amino acid sequence
encoded by each reading frame is indicated in the single-letter code below each codon.

Stop codons, of which there are three (50 -UAG-30 , 50 -UGA-30 , and
5 -UAA-30 ), define the end of the ORF and signal termination of polypeptide
0

synthesis. We can now fully appreciate the origin of the term open reading
frame. It is a contiguous stretch of codons “read” in a particular frame (as set
by the first codon) that is “open” to translation because it lacks a stop codon
(i.e., until the last codon in the ORF).
mRNAs contain at least one ORF. The number of ORFs per mRNA is dif-
ferent between eukaryotes and prokaryotes. Eukaryotic mRNAs almost
 Stop codons, of which there are three (50 -UAG-30 , 50 -UGA-30 , and

Messenger RNA: RBS

50 -UAA-30 ), define the end of the ORF and signal termination of polypeptide
synthesis. We can now fully appreciate the origin of the term open reading
frame. It is a contiguous stretch of codons “read” in a particular frame (as set
by the first codon) that is “open” to translation because it lacks a stop codon
(i.e., until the last codon in the ORF).
• Prokaryotic mRNAs have mRNAs a Ribosome-Binding Sitenumber
contain at least one ORF. The (RBS)ofthat
ORFs recruits
per mRNA theis dif-translational machinery.
ferent between eukaryotes and prokaryotes. Eukaryotic mRNAs almost
• This element is alsoalways
referred
containtoaas a Shine–Dalgarno
single sequence.
ORF. In contrast, prokaryotic mRNAs frequently
contain two or more ORFs and hence can encode multiple polypeptide
• The RBS is typicallychains.
located mRNAs3 – 9containing
bp ahead of theORFs
multiple start
arecodon.
known as polycistronic
mRNAs, and those encoding a single ORF are known as monocistronic
• It is complementary to a sequence
mRNAs. As we learned located near
in Chapter thepolycistronic
13, the 3’ end ofmRNAs
the 16S ribosomal
found in RNA (rRNA).
bacteria often encode proteins that perform related functions, such as
• The core of this region of the
different steps16S rRNA
in the has theof sequence
biosynthesis an amino acid 5’-CCUCCU-3’.
or nucleotide. TheNot surprisingly,
prokaryotic RBS arestructures
most often a subset
of typical of the
prokaryotic andsequence 5’-AGGAGG-3’.
eukaryotic mRNAs are shown in
Figure 15-2.

a
16S rRNA
3' U A G 5'
U CCUCC A CU

5' N N N N G G A G G N N N N N N N A U G N N 3'

stop RBS start stop RBS start stop

RBS start
3'
5' P P P
3–9 bases

5'PN N G
A N N A U G G N N 3' F I G U R E 15-2 Structure of messenger
 chains. mRNAs containing multiple ORFs are known as polycistronic
mRNAs, and those encoding a single ORF are known as monocistronic
mRNAs. As we learned in Chapter 13, the polycistronic mRNAs found in

Messenger RNA
bacteria often encode proteins that perform related functions, such as
different steps in the biosynthesis of an amino acid or nucleotide. The
structures of typical prokaryotic and eukaryotic mRNAs are shown in
Figure 15-2.

a
3' U G 5' 16S rRNA
U CCUCC AC UA

5' N N N N G G A G G N N N N N N N A U G N N 3'

stop RBS start stop RBS start stop

RBS start
3'
5' P P P
3–9 bases

5'PN N G
A N N A U G G N N 3'
• The extent of complementarity and the spacing between the RBS and the start
F I G U R codon has ofa messenger
E 15-2 Structure strong
RNA. (a) A polycistronic prokaryotic mes-
influence on how actively
5'
a particular
start ORF is translated.
AUG
stop
AAAAA 3' sage with three ORFs. Each ribosome-
n
binding site is indicated by a purple box
• Downstream ORF in a5'polycistronic
cap mRNA does not always need a strong RBS ⇒[Link]
labeled (b) A monocistronic eukaryotic

coupling. message. The 5 cap is indicated by a “ball”

0

at the end of the mRNA.

• In these cases, the start codon of the downstream ORF often overlaps the stop codon of the
upstream ORF (most often as the sequence 5’-AUGA-3’).
 50 -UAA-30 ), define the end of the ORF and signal termination of polypeptide
synthesis. We can now fully appreciate the origin of the term open reading
TA B L E 16-1 The Genetic Code frame. It is a contiguous stretch of codons “read” in a particular frame (as set
second position by the first codon) that is “open” to translation because it lacks a stop codon Translatio
U C A G
(i.e., until the last codon in the ORF).
5' mRNAs contain at least one ORF. The number of ORFs per mRNA is dif-
UUU UCU UAU UGU U
Phe Tyr Cys 3'
UUC UCC UAC UGC C
U Ser ferent
A U G A between
A A G C A A eukaryotes
U U U U C G U AandC U Gprokaryotes.
A A A G G U U GEukaryotic
G U G G C G C mRNAs
A C U U C Calmost
U G A
UUA UCA UAA* stop UGA* stop A
UUG
Leu
UCG UAG* stop UGG Trp G always
M Kcontain
A aI single
F ORF.
V In
L contrast,
K G prokaryotic
W W mRNAs
R T frequently
S stop

contain
5' two or more ORFs and hence can encode multiple polypeptide 3'
CUU CCU CAU
His
CGU U
chains. mRNAs containing multiple ORFs are known as polycistronic
A U G A A A G C A A U U U U C G U A C U G A A A G G U U G G U G G C G C A C U U C C U G A
CUC CCC CAC CGC C

third position (3' end)

mRNAs,
stop Kand Qthose F encoding
S Y astopsingle K ORF V are G known G A monocistronic
as L P
first position (5' end)

C Leu Pro Arg

CUA CCA CAA CGA A
CUG CCG CAG
Gln
CGG G mRNAs.
5'
As we learned in Chapter 13, the polycistronic mRNAs found3'in
bacteria
A U G A A Aoften encode
G C A A U U U U Cproteins
G U A C U G that
A A Aperform
G G U U G related
G U G G C functions,
G C A C U U C such
C U G Aas
AUU ACU AAU AGU U
AUC Ile ACC AAC
Asn
AGC
Ser
C
different E steps
S Nin theF biosynthesis
R T E of anR amino
L VacidA or nucleotide.
H F L The
A
AUA ACA
Thr
AAA AGA A
structures of typical prokaryotic and eukaryotic mRNAs are shown in
AUG† Met ACG AAG
Lys
AGG
Arg
G Figure 15-1 Three possible reading frames of the Escherichia coli trp leader sequence.
F I G U R E 15-2.
Start codons are shaded in green, and stop codons are shaded in red. The amino acid sequence
encoded by each reading frame is indicated in the single-letter code below each codon.
GUU GCU GAU GGU U
Asp
GUC GCC GAC GGC C
G Val Ala Gly
GUA GCA GAA GGA A
Glu a
16S rRNA
Stop3' Ucodons, of which thereG 5' are three (5 -UAG-3 , 5 -UGA-3 , and
GUG GCG GAG GGG G 0 0 0 0

5 -UAA-3U), C define U A
C U Cthe end
C of the ORF and signal termination of polypeptide
0 0
* Chain-terminating or “nonsense” codons. CA
† Also used in bacteria to specify the initiator formyl-Met-tRNAfMet. synthesis. We can now fully appreciate the origin of the term open reading
5' N NItNisNaG
frame. G A G G Nstretch
contiguous NNNN ofNcodons
NAUG N N 3'in a particular frame (as set
“read”
by the first codon) that is “open” to translation because it lacks a stop codon
(i.e., until the laststart
codon in the ORF).
stop RBS start stop RBS start stop
RBS
3'
5'mRNAs
P P Pcontain at least one ORF. The number of ORFs per mRNA is dif-
ferent between eukaryotes and prokaryotes. Eukaryotic mRNAs almost
O H H CH3 O H H CH3 3–9 bases
3' 3' always contain a single ORF. In contrast, prokaryotic mRNAs frequently
A O C C C CH2 A O C C C CH2 contain two or more ORFs and hence can encode multiple polypeptide
C C
C NH3 H CH3 C NH3 H CH3 chains. mRNAs containing multiple ORFs are known as polycistronic
A A
5' G C acceptor
leucine 5' G U
leucine mRNAs, and those encoding a single ORF are known as monocistronic
C G stem C G mRNAs. As we learned in Chapter 13, the polycistronic mRNAs found in
G C C G
A U G C b
bacteria often encode proteins that perform related functions, such as
A C A U different steps in the biosynthesis of an amino acid or nucleotide. The
C TψC stem
5'PN N G
G G C
D stem G C G C G
structures NN
Aof AUGG
typical N N 3'
prokaryotic and eukaryotic mRNAs are shown in
G
C C C C CC A U U G C C CU A FIGU
A U D AA Figure 15-2.
GGCGG A TψC D GGUGG A
Gm RNA.
GG GG G ψC loop AC GG GT ψC stop
 Challenges
1. Recognition of 20 distinct amino acids by the enzyme. Did DNA Pol do the same?

3. Absence of base pair complementarity.

6. How to know where to stop? How does stop codon work?

 Transfer RNA: The Adaptor
• tRNA molecules are adaptors between codons and the amino acids they specify.
• Each tRNA recognizes a specific amino acid and a particular codon, or codons, in the
mRNA (taking into account the redundancy).
• tRNA molecules are between 75 and 95 ribonucleotides in length.
• All tRNAs have certain features in common:
• all tRNAs end at the 3’ terminus with the sequence 5’-CCA-3’ (CCA-Adding Enzymes).
Consistent with this absolute conservation, this is the site that is attached to the
cognate amino acid.
 O N H

Transfer RNA: Unusual Bases ribose

pseudouridine

• All tRNAs have certain features in common: H

O
ribose
N3 4
• presence of several unusual bases in their primary structure 2
5
6

(achieved post-transcriptionally by enzymatic modification of

1
514 Chapter 15 O N

normal bases). uridine dihydrouridine

transcription
O cleotide cha
O
H H byHan isome
H 4
N
• For example, pseudouridine (𝛙U) is derived from uridine by an 5 ribose is sw
N3 4 3
5 H
2 6 2 6 position
H 5 (F
isomerization.
1 1
O N H O N H
by enzymati
5 and 6. Othe
• Likewise, dihydrouridine (D) is derived from uridine by enzymatic ribose ribose
and methylg
tion, but cel
reduction of the double bond between the carbons at positions 5 This observ
F I G U R E 15-3 A subset of modifie
and 6. pseudouridine
O
function. Fo
cleosides found in tRNA. Uridine an
an importan
uridine-related nucleotides are shown
H ribose
• Other unusual bases found in tRNA include hypoxanthine, N3 4
5
tRNAs Shar
thymine, and methylguanine O
2
1
6
Resembles
3'
a
N
A
• Cells lacking these modified bases show reduced rates of growth. dihydrouridine
As
C we saw i
C
complemen
5'
O that are held
H H no complem
N3 4 acceptor ar
5 H characteristi
2 6 H stranded reg
1 ψ
O N H
D loop (Fig. 15-4).U
 ture

FIGURE 15-3 A subset of modified nu- † T

Structure of Transfer RNA cleosides found in tRNA. Uridine and two
uridine-related nucleotides are shown.
a
t
e
s
• tRNAs share a common, conserved, secondary structure that 3' † T
resembles a cloverleaf: A
C u
C t
• The acceptor stem (site of attachment of the amino acid), is 5'
† T
formed by pairing between the 5’ and 3’ ends of the tRNA u
molecule. acceptor arm
† T
ψU loop t
• The 𝛙U loop (presence of pseudouridines in the loop). D loop c
• D loop (presence of dihydrouridines in the loop). o
† T
• The anticodon loop, contains the three-nucleotide-long variable a
sequence responsible for recognizing the codon by base pairing loop

with the mRNA. tRN

• The variable loop (varies in size from 3 to 21 bases) sits between anticodon loop
The
the anticodon loop and the 𝛙U loop. anticodon
is th
X-r
min
FIGURE 15-4 Cloverleaf representa-
tion of the secondary structure of tRNA.
loo
In this representation of a tRNA, the base ship
pairings between different parts of the acc
tRNA are indicated by the dotted red lines. tRN
 eo-
This
rge Structure of Transfer RNA in 3D Translation 515

rgy D loop form a second extended helix. These two extended helices align at a
right angle to each other, with the D loop and the CU loop coming together.
a
3'

Three kinds of interactions stabilize this L-shaped structure. First, the for-
the mation of the two extended regions of base pairing results in base-stacking
5'

s. • tRNAs have an L-shaped 3-dimensional structure. interactions similar to those seen in double-stranded DNA. Second, hydrogen
bonds are formed between bases in different helical regions that are brought
near each other in 3D space by the tertiary structure. These base–base interac-
c tions are generally unconventional (non-Watson–Crick) bonding. Finally,
ψU loop there are interactions between the bases and the sugar–phosphate backbone.
Translation 515

5' D loop form a second extended helix. These two extended helices align at a
ATTACHMENT OF AMINO ACIDS TO tRNA 3'

lanif eht fo snoiger deriap-esab eht fo noit

gniriap esab tsrif yb erutcurts lanif sti niatta

eht ot detaler si erutcurts ANRt lautca eht

etoN .ANRt a fo erutcurts dedlof lautca eht

eht neewteb noisrevnoC 5-51 E R U G I F

woh setartsulli margaid siht hguohtla taht

fo noitatneserper nobbiR )c( .ANRt dedlof

-acol eht gniwohs noitatneserper depahs-L

ton seod ANRt a ,noitatneserper faelrevolc

)b( .noitatneserper faelrevolC )a( .ANRt a

fo erutcurts D3 lautca eht dna faelrevolc

515
a

rotpecca
3' right angle to each other, with the D loop and the CU loop coming together.

mets nodocitna
wo

pool nodocitna

'3
Three kinds of interactions stabilize this L-shaped structure. First, the for-

mra
b 5'
mation
tRNAs AreofCharged
the two extended regions of of
by the Attachment base
anpairing
Aminoresults
Acid tointhe
base-stacking
acceptor

'3
.epahs L na otni gnidlof neht dna
ino

mets D

noitalsnarT
30 -Terminal
interactions similar to Nucleotide
those seen invia
double-stranded
a High-EnergyDNA.
AcylSecond,
Linkagehydrogen

'5
Adenosine 3'
D loop arm bonds are formed between bases in different helical regions that are brought

'5
5'
ase tRNA
nearmolecules
each othertoinwhich an amino acid is structure.
attached are saidbase–base
to be charged,

pool Uψ
3D space by the tertiary These interac-
and tRNAs
tions are that lack an
generally amino acid are
unconventional said to be uncharged.
(non-Watson–Crick) Charging
bonding. Finally,

'3
d to requires an interactions
there are acyl linkagebetween
betweenthethebases
carboxyl group
and the of the amino acid
sugar–phosphate and
backbone.

'5
the 20 - or 30 -hydroxyl group (see later discussion) of the adenosine nucleo-
D stem
ave variable
tide that protrudes from the acceptor stem at the 30 end of the tRNA. This

elbairav
acyl linkage is a high-energy bond because its hydrolysis results in a large

pool D
ATTACHMENT OFThis
AMINO ACIDSfor TOprotein
tRNAsynthesis: the energy
loop

pool
pal change in free energy. is significant

a
released when this acyl bond is broken is coupled to the formation of the
ysis anticodon stem peptide
tRNAs bonds that link amino
Are Charged by the acids to each of
Attachment other
an in polypeptide
Amino Acid tochains.
the
b

-rof eht ,tsriF .erutcurts depahs-L siht

onima eht ,esatehtnys eht morf desae

onima detalylyneda eht hcihw ni gni

onima eht hcihw ni noitalylyneda s

gnikcats-esab ni stluser gniriap esab

a ta ngila secileh dednetxe owt eseh

-nys eno rof nommocnu ton si ti ,)
yb deificeps era sdica onima tsom

sihT .ANRt htiw stcaer ,esatehtnys e

sisylordyh tneuqesbus eht si noitca

lapicnirp eht ,)9 retpahC ees( siseht
evah ew sA .enineda fo refsnart e
.ciremonom yllareneg era dna ANRt
semyzne I ssalC .)1-51 elbaT( sesate

-oelcun enisoneda eht fo )noissucsi

-caretni esab – esab esehT .erutcurts yr

thguorb era taht snoiger lacileh tnere
-pyt era dna ANRt eht fo HO- 03 eht ot

eht ,noitalylyneda fo tluser a sA .e

esaeler tnatimocnoc eht htiw detalyl

eht fo noitamrof eht ot delpuoc si n

ygrene eht :sisehtnys nietorp rof tna

,yllaniF .gnidnob )kcirC – nostaW-no

negordyh ,dnoceS .AND dednarts-elb

,elgnis a yb ANRt etairporppa eht ot

aiv ANRt eht fo dne 03 eht ot dica oni

lyrohpsohp eht ot denioj si dica on

ni dnob retse ygrene-hgih a aiv di

ot desoppo sa ,PMA fo refsnart ot s

owt ni ANRt a ot dica onima na h

egral a ni stluser sisylordyh sti esua

dna dica onima eht fo puorg lyxobra

,degrahc eb ot dias era dehcatta si di

ANRt eht neewteb gnilpuoc laitini eh

sihT .ANRt eht fo dne 03 eht ta met

gnigrahC .degrahcnu eb ot dias er

.rehtegot gnimoc pool UC eht dna p

.enobkcab etahpsohp – ragus eht dna
30 -Terminal Adenosine Nucleotide via a High-Energy Acyl Linkage c 3'

.sniahc editpepylop ni rehto hcae

.HO- 02 eht dna HO- 03 eht ta tnemh

ψU loop
the Aminoacyl-tRNA Synthetases Charge tRNAs in Two Steps 5'

egakniL lycA ygrenE-hgiH a

tRNA molecules to which an amino acid is attached are said to be charged, 5'
anticodon loop
dicA onimA elgniS a sehcat

eht ot dicA onimA na fo tn

be uncharged. 3'
d in Alland tRNAs that lack
aminoacyl-tRNA
requires an acyl linkage
an aminoattach
synthetases
between
acid are
the
said toacid
an amino
carboxyl group
to a tRNA in
of
enzymatic steps (Fig. 15-6). Step one is adenylylation in which the amino the amino
Charging
two
acid and acceptor
arm
20 - orwith
thereacts 30 -hydroxyl group (see later discussion) ofconcomitant
the adenosine nucleo- D loop
oryl acid ATP to become adenylylated with the release

spetS owT ni sANRt

tide that protrudes from the acceptor stem at the 30
end
of pyrophosphate. Adenylylation refers to transfer of AMP, as opposed to of the tRNA. This
acyl linkage is a high-energy bond because its hydrolysis results in a large D stem
ino adenylation, which would indicate the transfer of adenine. As we have
change
seen in theincase
freeofenergy. This is significant
polynucleotide synthesis for(seeprotein
Chapter synthesis:
variable
the energy loop
9), the principal
FIGURE 15-5 Conversion between the

ANRt OT S
released when thisadenylylation
acyl bond is broken is is
coupled to the formation of the anticodon stem
This driving
peptide
force
bonds
for the
that link amino
reaction
acids to each
the subsequent
other in
of pyrophosphate by pyrophosphatase. As a result of adenylylation, the
hydrolysis
polypeptide chains.
cloverleaf and the actual 3D structure of
.PMA f

anticodon loop
via a tRNA. (a) Cloverleaf representation. (b)
amino acid is attached to adenylic acid via a high-energy ester bond in
which the carbonyl group
Aminoacyl-tRNA of the Charge
Synthetases amino acid
tRNAs is joined
in TwotoSteps
the phosphoryl
c
ψU loop
 Structure of Transfer RNA
ATTACHMENT OF AMINO ACIDS TO tRNA
b
tRNAs Are Charged by the Attachment of an Amino Acid to the
30 -Terminal Adenosine Nucleotide via a High-Energy Acyl Linkage
• Three kinds of interactions stabilize this L-shaped structure. 5'
3'

tRNA molecules to which an amino acid is attached are said to be charged,

1. two extendedandregions
tRNAs of thatbase
lack anpairing results
amino acid in tobase-stacking
are said be uncharged. Charging
interactions. requires an acyl linkage between the carboxyl group of the amino acid and
the 2 - or 30 -hydroxyl group (see later discussion) of the adenosine nucleo-
0

2. hydrogen bondstideare formedfrom

that protrudes between bases
the acceptor stemin different
at the 30 end of helical
the tRNA. This
acyl linkage is a high-energy bond because its hydrolysis results in a large
regions that are brought
change near each
in free energy. This is other in for
significant 3Dprotein
spacesynthesis: the energy
released when this acyl bond is broken is coupled to the formation of the
3. there are interactions
peptide bonds between the bases
that link amino andother
acids to each theinsugar–
polypeptide chains.
phosphate backbone. c
ψU loop
Aminoacyl-tRNA Synthetases Charge tRNAs in Two Steps
5'
All aminoacyl-tRNA synthetases attach an amino acid to a tRNA in two 3'
enzymatic steps (Fig. 15-6). Step one is adenylylation in which the amino acceptor
D loop arm
acid reacts with ATP to become adenylylated with the concomitant release
of pyrophosphate. Adenylylation refers to transfer of AMP, as opposed to
D stem
adenylation, which would indicate the transfer of adenine. As we have variable
seen in the case of polynucleotide synthesis (see Chapter 9), the principal loop
driving force for the adenylylation reaction is the subsequent hydrolysis anticodon stem
of pyrophosphate by pyrophosphatase. As a result of adenylylation, the
amino acid is attached to adenylic acid via a high-energy ester bond in anticodon loop
which the carbonyl group of the amino acid is joined to the phosphoryl
group of AMP. Step two is tRNA charging in which the adenylylated amino
FIGURE 15-5 Conversion between the
acid, which remains tightly bound to the synthetase, reacts with tRNA. This
cloverleaf and the actual 3D structure of
 tures f
† The
Attachment Of Amino Acids To tRNA
FIGURE 15-3 A subset of modified nu-
cleosides found in tRNA. Uridine and two ami
uridine-related nucleotides are shown. tRN
ecu
stem
• The problem of attaching amino acids to nucleotides remain. 3' † The
Where is base pairing? A
C unu
C the
• A tRNA molecule without an amino acid attached is said to be 5'
† The
uncharged. urid
acceptor arm
• tRNAs are charged by the attachment of an amino acid to the † The
ψU loop thre
3’-terminal adenosine nucleotide D loop cod
on
• Takes place via a high-energy acyl linkage between the
† The
carboxyl group of the amino acid and the 2’- or 3’- hydroxyl as i
variable
group of the adenosine nucleotide that protrudes from the loop
acceptor stem.
tRNA
• Catalyzed by specialized enzyme called Aminoacyl-tRNA anticodon loop
The cl
Synthetase. is the
anticodon
X-ray
• Energy released when this acyl bond is broken is used for the minus
formation of the peptide bonds. FIGURE 15-4 Cloverleaf representa-
tion of the secondary structure of tRNA.
loop i
In this representation of a tRNA, the base ship o
pairings between different parts of the accep
tRNA are indicated by the dotted red lines. tRNA
 Attachment Of Amino Acids To tRNA
516 Chapter 15

516 Chapter 15
a
FIGURE 15-6 The two steps of amino-
• Aminoacyl-tRNA Synthetases charge tRNAs in two steps:
acyl-tRNA charging. (a) Adenylylation of
F I G U(b)
amino acid. 15-6 The
R ETransfer twoadenylylated
of the steps of amino-
a
R O O O O A
amino acyl-tRNA
acid to tRNA. charging. (a) Adenylylation
The process shown is of
NH2 CR CO O
–
+ –
O O P OO P OO P O A
• Adenylylation: amino acid reacts with ATP to become
amino acid. (b) Transfer of the adenylylated
for a class II tRNA synthetase (which attaches
amino acid to tRNA. The process shown is
the amino acid to the 30 -OH).
for a class II tRNA synthetase (which attaches NH2 HC C O
–
+ –
O P O–O P OO
– P O–
O
adenylylated with the concomitant release of the amino acid to the 30 -OH).
amino
H acid O– O– ATPO–
OHOH
OHOH

pyrophosphate.
amino acid ATP

• the principal driving force for the adenylylation reaction R

R
O
O
O
O A
A
O
O
O
O

is the subsequent hydrolysis of pyrophosphate by NH2

NH2
C
C
C
C
O
O
P
P O
O
++ –
O
–
O
P
P
O
O
P
P
O–
O–

pyrophosphatase. HH
adenylylated
adenylylated
O–
O
–

OHOH
OHOH
O– O–
pyrophosphate
pyrophosphate
O– O–

amino acid
amino acid

• As a result of adenylylation, the amino acid is attached bb adenylylated amino

adenylylated aminoacid
acid
to adenylic acid via a high-energy ester bond between RR O
O O
O A A

the carbonyl group of the amino acid and the NH

NH2 2 C
C
C
C O
O
P
P
O
O
tRNA
tRNA

phosphoryl group of AMP. H

H O
O–
–

OHOH
OHOH
C
AC

• tRNA charging: adenylylated amino acid, which remains

3' -OH C
AC
3' -OH

tightly bound to the synthetase, reacts with tRNA.

• This reaction results in the transfer of the amino acid to high-energy
+ –
O
O

P O
O
A

A
the 3’ end of the tRNA and the release of AMP. NH2 RC
R

C
O
O bond
high-energy
O ACC
bond + –
OO
–
P O
OH OH
H O–
NH2 C Ccharged
O tRNA
ACC
AMP OH OH
H
charged tRNA AMP
 How many Adaptors?
• Each of the 20 amino acids is attached to the appropriate tRNA by a single, dedicated tRNA
synthetase.
• Because most amino acids are specified by more than one codon, each synthetase recognize and
charge more than one tRNA (known as iso-accepting tRNAs).

• Most organisms have 20 different tRNA synthetases, although not always:

• Some bacteria lack a synthetase for charging the tRNA for glutamine (tRNAGln) with its cognate
amino acid.
• Instead, a single species of aminoacyl-tRNA synthetase charges tRNAGln as well as tRNAGlu with
glutamate.
• A second enzyme then converts (by amination) the glutamate of the charged tRNAGln molecules
to glutamine. That is, Glu-tRNAGln is aminated to Gln-tRNAGln .
• An aminoacyl-tRNA synthetase can never attach more than one kind of amino acid to a given
tRNA: one-to-one correspondence between amino acid and aminoacyl-tRNA synthetase.
 sequence could recognize several different codons. Cases were also discov-
ered in which an anticodon base was not one of the four regular ones, but a
fifth base, inosine. Like all the other minor tRNA bases, inosine arises

Does it mean there are 61 tRNAs? through enzymatic modification of a base present in an otherwise com-
pleted tRNA chain. The base from which it is derived is adenine, whose
carbon 6 is deaminated to give the 6-keto group of inosine. (Inosine is
actually a nucleoside composed of ribose and the base hypoxanthine,
but it has come to be referred to as a base in common usage and we do
so here.)
In 1966, Francis Crick devised the wobble concept to explain these obser- TA BL E16-2 Pairing Combinations with
• No, there are less. Because of the wobble concept. vations. It states that the base at the 50 end of the anticodon is not as spatially
confined as the other two, allowing it to form hydrogen bonds with any of
the Wobble Concept

Base in Anticodon Base in Codon

several bases located at the 30 end of a codon. Not all combinations are pos-
• It states that the base at the 5’ end of the anticodon is not as sible, with pairing restricted to those shown in Table 16-2. For example, U at
the wobble position can pair with either adenine or guanine, while I can pair
G
C
U or C
G

spatially confined as the other two, allowing it to form with U, C, or A (Fig. 16-2). The pairings permitted by the wobble rules
A
U
U
A or G
are those that give ribose –ribose distances close to that of the standard
hydrogen bonds with any of several bases located at the 3’ A:U or G:C base pairs. Purine –purine (with the exception of I:A pairs) or
I A, U, or C

end of a codon. 576 Chapter 16

• Permitted pairings are those that give ribose–ribose

a
distances anticodon codon
H
b
3' 5' 3' 5'
anticodon arm of tRNA

close to that of the standard A:U or G:C base pairs H N O H N H anticodon

TA B L E 16-1 The Genetic Code
N UC U UC U mRNA chain
second position N H N H
ribose
U C A G N N 5' A GA 3' 5' A GG 3'
UUU UCU UAU UGU U H O ribose
Phe Tyr Cys codon
UUC UCC UAC UGC C inosine–cytosine U in the first (5' ) anticodon
U Ser
UUA UCA UAA* stop UGA* stop A
H position can pair with A or G
Leu O
UUG UCG UAG* stop UGG Trp G

CUU CCU CAU CGU U H N O H N H c

His 3' 5' 3' 5' 3' 5'
CUC CCC CAC CGC C
third position (3' end)
N
first position (5' end)

C Leu Pro Arg

CUA CCA CAA CGA A N N H O ribose
Gln
CUG CCG CAG CGG G ribose
N
AUU ACU AAU AGU U H
Asn Ser CC I CC I CC I
AUC Ile ACC AAC AGC C inosine–uracil
A Thr
AUA ACA AAA AGA A 5' G GU 3' 5' G GC 3' 5' GGA
Lys Arg H
AUG† Met ACG AAG AGG G

H N O H N N H I in the first (5' ) anticodon

GUU GCU GAU GGU U position can pair with U, C, or A
Asp
GUC GCC GAC GGC C
G Val Ala Gly N N N
GUA GCA GAA GGA A N H
Glu ribose ribose
GUG GCG GAG GGG G N N
* Chain-terminating or “nonsense” codons. H H
inosine–adenine
 Challenges
1. Recognition of 20 distinct amino acids by the enzyme. Did DNA Pol do the same?

3. Absence of base pair complementarity.

9. How does tRNA recognize the correct Amino Acid?

 amino acid, and the superscript identifies the type of codon the tRNA recog-
nizes). The presence of this second enzyme removes the need for a gluta-

Recognition of correct tRNA

mine tRNA synthetase. Nevertheless, an aminoacyl-tRNA synthetase can
never attach more than one kind of amino acid to a given tRNA.

tRNA Synthetases Recognize Unique Structural Features

of Cognate tRNAs
• aa-tRNA Synthetases must recognize the correct set of tRNAs for a particular amino acid.
As we can see from the above considerations, aminoacyl-tRNA synthetases
face two important challenges: they must recognize the correct set of tRNAs
• What features of the tRNA molecule enable
for a particular a synthetase
amino acid, toalldiscriminate
and they must charge of these iso-accepting the correct set of iso-
tRNAs with the correct amino acid. Both processes must be performed
accepting tRNAs from the tRNAswithfor
highthe other 19 amino acids.
fidelity.
Let us first consider the specificity of tRNA recognition: what features of
• Specificity determinants are clustered at two distant sites on the molecule: the acceptor stem the tRNA molecule enable a synthetase to discriminate the correct set of iso-
accepting tRNAs from the tRNAs for the other 19 amino acids? Genetic, bio-
and the anticodon loop. chemical, and X-ray crystallographic evidence indicates that the specificity
determinants are clustered at two distant sites on the molecule: the acceptor
stem and the anticodon loop (Fig. 15-7). The acceptor stem is an especially
• Because each amino acid is usually specified by more than one codon, dependence on anticodon important determinant for the specificity of tRNA synthetase recognition. In

recognition cannot be too strong. some cases, changing a single base in the acceptor stem (known518 as the Chapter 15

TA B L E 16-1 The Genetic Code

second position discriminator base) is suffic
U C A G acceptor discriminator 3' acceptor tRNA from one synthetase to
stem base end
UUU UCU UAU
Tyr
UGU
Cys
U quently contributes to discri
Phe
U
UUC UCC
Ser
UAC UGC C for example, makes numerou
UUA UCA UAA* stop UGA* stop A
Leu the anticodon loop, includin
UUG UCG UAG* stop UGG Trp G
One might expect that th
CUU CCU CAU CGU U tion by tRNA synthetases b
CUC CCC CAC
His
CGC C Aminoacyl
tRNA—the anticodon dictat
third position (3' end)
first position (5' end)

C Leu Pro Arg

CUA CCA CAA CGA A for incorporating into the g
CUG CCG CAG
Gln
CGG G -AMP
each amino acid is usually s
AUU ACU AAU AGU U of the anticodon cannot be
Asn Ser
AUC Ile ACC AAC AGC C acid serine is specified by s
A Thr
AUA ACA AAA AGA A which are completely differe
Lys Arg
AUG† Met ACG AAG AGG G anticodon stem ine necessarily have a varie
GUU GCU GAU GGU U
easily recognized by a singl
GUC GCC GAC
Asp
GGC C tRNAs, the synthetase for se
G Val Ala Gly anticodon
GUA GCA GAA GGA A
anticodon loop of the anticodon.
Glu
GUG GCG GAG GGG G

* Chain-terminating or “nonsense” codons.

FIGURE 15-7 Structure of tRNA: elements required for aminoacyl synthetase recognition.
F I G U R E 15-8 Cocrystal structure of Aminoacyl-tRNA Formation
† Also used in bacteria to specify the initiator formyl-Met-tRNAfMet.
glut-aminyl aminoacyl-tRNA synthetase
 the anticodon loop, incl
cases, it is easy to understand
pared with MolScript, BobScript, and how this high
One might expect tha
Raster3D. ple, the amino acids cysteine and
tion by tRNA tryptoph
synthetas

Recognition of the correct Amino Acid shape, and chemical groups.

acids tyrosine and phenylalanine
tRNA—the
Even in the cad
each amino
anticodon
for incorporating into t
(seeacidFig. 1
is usua
ing a strong and energetically of thefavorable
anticodon canno hyd
acid serine is specified
moiety of the former but not whichthe are latter
completely allo
di

Selecting the correct amino acid is moreadaunting than the challenge(tyrosyl-tRNA

inIt isrecognizing synthetase) to discriminate efv
the easily
ine necessarily have a
• more challenging totRNAs,
recognized by a s
understand thefo
OH
appropriate tRNA: small size and similarity.
the synthetase
which differ by only a single methylene
of the anticodon. gro

synthetase can sterically exclude isoleuc

• Despite this challenge, the frequency of mischarging is very low; typically, F I G U R E less
because than
15-8 Cocrystal
glut-aminylisoleucine is1larger
structure
aminoacyl-tRNA synthetase
in
of 1000
Aminoacyl-tRNA Form
than valine. In co
tRNAs into the catalytic pocket of The
with tRNA . Enzyme (gray); tRNA Gln

( purple). The yellow, red, and green mole-

thechallenge
isoleucyl-tR
Gln
faced by a
amino acid is perhaps e
CH2 CH2
amino acids will
cule is glutaminyl-AMP. Notefit
theinto the isoleucyl-tRNA
proximity
faces in recognizing the
• For certain amino acids, like for cysteine and tryptophan, the difference in size, shape,
of this molecule to the 3 end of the tRNA
site, interactions with
and the points of contact between the the and
0

extra
Despite
methylene
the relatively
this
small size
challenge,
g
t
chemical groups is substantial. C H C H antRNA
extra – 2 to – 3 kcal/mol less
and the synthetase. (Rath V.L. et al.
1998. Structure 6: 439– 449.) Image pre-
of than
free1 in energy
1000 tRNA (s
H 2N COOH H2N COOH described in Chapter
pared with MolScript, BobScript, 3,andeven this
cases, it isrelatively
easy to under

Others like isoleucine and valine, differ by only

tyrosinea single phenylalanine
methylene group.
Raster3D. ple, the amino acids cy
• will make binding to isoleucine shape, and!100-fol
chemical gro
valine if the two amino acids are present
acids tyrosine and phen
ing a strong and energe
b
valine would be attached tomoiety isoleucine tRN
of the former bu
this is an unacceptably high(tyrosyl-tRNA rate of error. A
synthetas
CH3 a
It is more challengin
frequency of misincorporation
OH
is ,0.1%.
which differ by only a s
CH2 CH3 CH3 CH3 fidelity achieved? synthetase can sterical
CH CH because isoleucine is lar
into the catalytic pocke
amino acids will fit into
C H C H
CH2 CH2
Some Aminoacyl-tRNA Synthetases Use asite, interactions with th
H 2N COOH H2N COOH C C an extra – 2 to – 3 kcal/m
to Charge tRNAs with High Accuracy
H 2N
H
COOH H2N
H
COOH described in Chapter 3,
isoleucine valine tyrosine phenylalanine will make binding to i
One common mechanism tovaline increase theamino
if the two fid
valine would be attache
FIGURE 15-9 Distinguishing features thetase
b is to proofread the products of the ch
this is an unacceptably
CH3
of similar amino acids. for DNA
CH2 CH3
polymerases
CH3 CH3
in Chapter
frequency [Link] exam
misincorp
fidelity achieved?
CH CH
 C H C H
H 2N COOH H2N COOH

Recognition of the correct Amino Acid tyrosine phenylalanine

b
CH3
• Valyl-tRNA synthetase can sterically exclude isoleucine from its catalytic CH2 CH3 CH3 CH3
pocket (larger). CH CH

• This is not true for isoleucyl-tRNA synthetase. Valine can slip easily into its C H C H
H 2N COOH H2N COOH
catalytic pocket. isoleucine valine

• However, interactions with the extra methylene group on isoleucine will FIGURE 15-9 Distinguishing features
provide an extra –2 to –3 kcal/mol of free energy: gives 1 in 100 accuracy of similar amino acids.

• To further increase the fidelity, aminoacyl-tRNA synthetase proofreads the

products of the charging reaction:
• e.g. isoleucyl-tRNA synthetase has an editing pocket that allows AMP-valine
to fit into, where it is hydrolyzed and released as free valine and AMP. In
contrast, AMP-isoleucine is too large to fit and is therefore not subject to
hydrolysis.
• ⇒ Discriminates twice!
 Accuracy of aminoacyl-tRNA linkage

• Ribosome “blindly” accepts any charged tRNA that shows a proper codon – anticodon
interaction, whether or not the tRNA is charged with the correct amino acid.
• This is shown through genetic experiments with mutant tRNA that carries a nucleotide
substitution in the anticodon.
• Shown also through biochemical experiments where cysteine attached to cysteine-
tRNACys is converted to an alanine by chemical reduction to give alanine-tRNACys .
 The Ribosome

• It has a lot of task in hand:

• To bind mRNA.
• To read information in mRNA, one codon at a time.
• To bring in the amino acids to its catalytic centre based on the codons present there.
• To stitch these amino acids together.
 Prokaryotic Ribosome
522 Chapter 15

• The Ribosome (70S) Is composed of a large

5.8S rRNA 5S rRNA
and a small subunit. (160 (120
nucleotides) 50S nucleotides)
• The large subunit (50S) contains
60S the peptidyl prokaryotic
(MW= 1,600,000)
5S rRNA 23S rRNA
transferase center, which is responsible for
(MW= 2,800,000)
(120 ribosome (2,900
the formation
eukaryoticof peptide bonds. nucleotides) 70S nucleotides)
ribosome (MW= 2,500,000)
• The small 80S
subunit (30S) contains the 28S rRNA
(4,700 ~34 proteins
decoding center in which charged tRNAs nucleotides)
(MW= 4,200,000) read
the codon units of the mRNA.
49 proteins 30S 16S rRNA
• Although there are many ribosomal proteins (MW= 900,000) (1,540
nucleotides)
in number, more than two-thirds of the mass
40S 18S rRNA
of the prokaryotic ribosome is RNA.
(MW= 1,400,000) (1,900
21 proteins

nucleotides)

MW of a.a ~ 110 Da; MW of nucleotide ~ 330 Da

~33 proteins

FIGURE 15-13 Composition of the prokaryotic and eukaryotic ribosomes. The rRNA and
protein composition of the different subunits are indicated. The length of the rRNA and the
number of ribosomal proteins are indicated for each subunit.
 (known as ribosomal RNAs or rRNAs), and many ribosomal proteins (Fig.
15-13). Svedberg units are once again used to distinguish among the rRNAs.

Prokaryotic Ribosome Thus, in bacteria, the 50S subunit contains a 5S rRNA and a 23S rRNA,
whereas the 30S subunit contains a single 16S rRNA. Although there are
far more ribosomal proteins than rRNAs in each subunit, more than two-
thirds of the mass of the prokaryotic ribosome is RNA. This is true because

centrifuge
• S — Svedberg — unit used to measure
sedimentation velocity in ultracentrifuge.
• The larger the S value the faster the
sedimentation velocity and the larger the
molecule.
30S 50S 70S
• Note that 70S is less than the sum of 50S and ribosomal subunits
30S!
FIGURE 15-12 Sedimentation by ultracentrifugation separates the bacterial ribosome
• Sedimentation velocity is determined by both subunits from the full ribosome.

shape and size and is not an exact measure of

mass.
 Eukaryotic Ribosome

522 Chapter 15
• The eukaryotic ribosome is somewhat larger,
composed of 60S and 40S subunits, which together 5.8S rRNA
(160
form an 80S ribosome. 60S
nucleotides)

5S rRNA prokaryo
(MW= 2,800,000)
(120 ribosom
eukaryotic nucleotides) 70S
ribosome (MW= 2,500
80S 28S rRNA
(MW= 4,200,000) (4,700
nucleotides)

49 proteins

40S 18S rRNA

(MW= 1,400,000) (1,900
nucleotides)

~33 proteins

FIGURE 15-13 Composition of the

protein composition of the different su
number of ribosomal proteins are indicat
 mence translation of the mRNA as it emerges from the RNA polymerase.
This situation allows the ribosome to proceed in tandem with the RNA poly-
merase as it elongates the transcript (Fig. 15-11). Recall that the 50 end of an
RNA is synthesized first, and thus the ribosome, which begins translation at

The Ribosome the 50 end of the mRNA, can start translating a nascent transcript as soon as it
emerges from the RNA polymerase. Interestingly, there are several instances

RNAP

• Overall molecular mass of > 2.5 MDa.

• Rate of translation only ~ 20 amino acids/sec (60
nucleotides/sec).
5'
• In Prokaryotes, the ribosome commences translation in
tandem with transcription.
• Eukaryotic transcription occurs in the nucleus, whereas
translation occurs in the cytoplasm.
• Eukaryotic translation speed: ~ 2-4 amino acids/ sec. 5'

FIGURE 15-11 Prokaryotic RNA polymerase and ribosomes at work on the same mRNA.
 nery are located in the same compartment. Thus, the ribosome can com-
mence translation of the mRNA as it emerges from the RNA polymerase.
F I G U R E 15-14 Overview of the events of translation: the ribosome cycle. This situation allows the ribosome to proceed in tandem with the RNA poly-
merase as it elongates the transcript (Fig. 15-11). Recall that the 50 end of an
The Ribosome
80 nucleotides of mRNA. Still, even a small ORF of 1000 bases (which would
RNA is synthesized first, and thus the ribosome, which begins translation at
the 50 end of the mRNA, can start translating a nascent transcript as soon as it
encode a protein of !35 kDa) can bind more than 10 ribosomes and therefore emerges from the RNA polymerase. Interestingly, there are several instances
direct the simultaneous synthesis of multiple polypeptides.
The ability of multiple ribosomes to function on a single mRNA explains RNAP
• Each mRNA can be translated simultaneously by
the relatively limited abundance of mRNA in the cell (typically 1% –5% of
total RNA). If an mRNA could be translated by only one ribosome at a time,
multiple ribosomes (polysome).
then as few as 10% of the ribosomes would be engaged in protein synthesis
in a typical cell. Instead, the association of multiple ribosomes with each
• A single ribosome contacts ~ 30 nucleotides.
mRNA ensures that the majority of the ribosomes are engaged in translation
at any given time.

• But large size of the ribosome ⇒ a density of one 5'

New Amino Acids Are Attached to the Carboxyl Terminus of the

ribosomeGrowing
for every 80Chain
Polypeptide nucleotides.
• Still, evenAsawesmall ORF of 1000 bases can bind
know, both polynucleotide and polypeptide chains have intrinsic
polarities. Thus, for each of these molecules, we can ask which end of the
more than 10is synthesized
chain ribosomes. first. We learned in Chapters 9 and 13 that DNA and
RNA are synthesized by adding each new nucleotide triphosphate to the
30 end of the growing polynucleotide chain (often referred to as synthesis
in the 50 !30 direction). 5'

FIGURE 15-11 Prokaryotic RNA polymerase and ribosomes at work on the same mRNA.
growing
polypeptide

5'
3'
mRNA start (AUG)
stop (UAG)

FIGURE 15-15 A polyribosome.

 we discussed above i
Acids to tRNAs), the

Chapter 15
Formation of The Polypeptide Chain group of the amino a
manner (at its 30 end
chain. The bond betw
ken during the form
6 Chapter 15
a between the peptidy
15-6 The two steps of amino-
charging. (a) Adenylylation of
high-energy acyl bond
as the growing chai
a O O O polypeptide chain
G
d.U(b) 15-6 The
R ETransfer twoadenylylated
of the steps of amino- R O A
dl-tRNA charging.
to tRNA. (a) Adenylylation
The process shown is of H O attached to the amin
Ino acid. (b) Transfer of the adenylylated
tRNA synthetase (which attaches NH2 CR CO O
–
+ –
O O P OO P OO P O A H H
To catalyze pepti
no acid to tRNA. The process shown is N C C
acid to the 30 -OH).
a class II tRNA synthetase (which attaches NH2 HC C O
–
+ –
O P O–O P OO
– P O–
O N C R H 2N C R
brought into close pr
H R
amino acid to the 30 -OH). OHOH
amino
H acid O– O– ATPO– H C O C O ing allows the amino
OHOH
amino acid ATP
O O attack the carbonyl g
to the peptidyl-tRNA
of a new peptide bon
R O O A O O
R O O A O O the release of the po
++ There are two conse
–
NH2 C C O P O – O P O P O–
NH2 C C O P O O P O P O–
HH O–
O
–
O– O– O– O– this mechanism of pe
adenylylated
adenylylated OHOH
OHOH pyrophosphate
pyrophosphate
aminoacyl-tRNA
of the protein be sy
amino acid
amino acid peptidyl-tRNA
growing polypeptid
bb adenylylated amino
adenylylated aminoacid
acid
aminoacyl-tRNA. Fo
O
is called the peptidy
RR O
O O A A
Interestingly, pep
NH C C O P O tRNA H O
NH2 2 C C O P O tRNA ous hydrolysis of a n
–
R O H
H O N C C
H O– OHOH N C C N C R formation is driven b
OHOH H R
ing polypeptide chai
C
AC H H H C O
3' -OH
3' -OH AC
C
O
the tRNA synthetas
tRNA. The charging
OH
Thus, the energy for
O A of ATP that was hyd
R O
high-energy
bond + –
O P O
O A

NH2 RC C
O
high-energy
O ACC
bond + –
OO
–
P O
Ribosomal RNAs A
OH OH
–
NH2 C
H
Ccharged
O tRNA
ACC
O
AMP
Determinants of the
OH OH
H Although the ribosom
charged tRNA AMP FIGURE 15-16 The peptidyl transfer-
ase reaction. years ago, the determ
 we discussed above i
Acids to tRNAs), the

Formation of The Polypeptide Chain group of the amino a

manner (at its 30 end
chain. The bond betw
ken during the form
between the peptidy
high-energy acyl bond
• New amino acids are attached to the carboxyl terminus of polypeptide chain as the growing chai
attached to the amin
the growing polypeptide chain. N
H

C
O

C
H H
To catalyze pepti
N H 2N C R
C R
brought into close pr
• Both the growing chain and the incoming amino acid are H R
H C O C O ing allows the amino
attached to tRNAs (an aminoacyl-tRNA and a peptidyl- O O attack the carbonyl g
to the peptidyl-tRNA
tRNA) ⇒ the growing polypeptide is continuously attached of a new peptide bon
to a tRNA. the release of the po
There are two conse
• The bond between the aminoacyl-tRNA and the amino acid this mechanism of pe
of the protein be sy
is not broken. peptidyl-tRNA aminoacyl-tRNA
growing polypeptid
aminoacyl-tRNA. Fo
• Instead, the bond between the peptidyl-tRNA and the is called the peptidy
Interestingly, pep
attached polypeptide chain is broken as the growing chain H O
R O H ous hydrolysis of a n
is attached to the amino group of the amino acid attached N C C
N C C N C R formation is driven b
to the aminoacyl-tRNA to form a new peptide bond ing polypeptide chai
H R
H H H C O
the tRNA synthetas
(peptidyl transferase reaction). OH
O
tRNA. The charging
Thus, the energy for
• To catalyze peptide-bond formation, the 3’ ends of these of ATP that was hyd
two tRNAs are brought into close proximity by the
ribosome. Ribosomal RNAs A
Determinants of the

FIGURE 15-16 The peptidyl transfer- Although the ribosom

ase reaction. years ago, the determ
 tions of the ribosome. The most obvious example of this is the demonstration
that the peptidyl transferase center is composed almost entirely of RNA, as
Ribosome has three Binding Sites for tRNA
discussed in detail later. RNA also plays a central role in the function of
the small subunit of the ribosome. The anticodon loops of the charged tRNAs
and the codons of the mRNA contact the 16S rRNA, not the ribosomal pro-
teins of the small subunit.
A further indication of the importance of RNA in the structure and func-
• Needs to bind at least two tRNAs simultaneously.
tion of the ribosome is that most ribosomal proteins are on the periphery of
• Contains three
the ribosome, not tRNA-binding
in its interior ([Link], called
15-17; see the A-, P-,
also Structural and E-sites.
Tutorial 15-1).
The core functional domains of the ribosome (the peptidyl transferase cen-
• Each tRNA-binding
ter and site isare
the decoding center) formed at the
composed interface
either entirely orbetween
mostly fromthe large and the small
subunits of the
RNA. Portions ribosome.
of some ribosomal proteins do reach into the core of the sub-
units, where their function seems to be to stabilize the tightly packed rRNAs
by shielding the negative charges of their sugar –phosphate backbones.
Indeed, it is likely that the contemporary ribosome evolved from a primitive
protein-synthesizing machine that was composed entirely of RNA and that E P A
the ribosomal proteins were added to enhance the function of this primor-
dial RNA machine. mRNA
5' 3'

The Ribosome Has Three Binding Sites for tRNA FIGURE 15-18 The ribosome has
three tRNA-binding sites. The schematic il-
To perform the peptidyl transferase reaction, the ribosome must be able to lustration of the ribosome shows the three
bind at least two tRNAs simultaneously. In fact, the ribosome contains three binding sites (E, P, and A) that each spans
tRNA-binding sites, called the A-, P-, and E-sites (Figs. 15-18 and 15-19). The the two subunits.
 Ribosome Has Three Binding Sites for tRNA
526 Chapter 15

a b

• The E-, P-, and A-site tRNAs are shown in

yellow, red, and green, respectively.

Decoding center
c d

Peptidyl transferase center

50S subunit
30S subunit
FIGURE 15-19 Views of the 3D structure of the ribosome including three bound tRNAs.
 molecular base-pairing interactions that may have formed in the mRNA. In views of the structure of the
between the two channels is a region that is accessible to tRNAs and where tRNAs are shown as they are fou
bosome. For clarity, the ribos
adjacent codons can bind to the aminoacyl-tRNA and peptidyl-tRNA in the

Entry and Exit of mRNA, tRNAs and growing polypeptide

shown. The E-, P-, and A-site
A- and P-sites, respectively. Interestingly, there is a pronounced kink in the shown in yellow, red, and green
mRNA between the two codons that facilitates maintenance of the correct ly, and the mRNA is shown in bl
reading frame (Fig. 15-20). This kink places the vacant A-site codon in a dis- bases involved in the codon–an

through the Ribosome tinctive position that ensures that the incoming aminoacyl-tRNA does not
have access to bases immediately adjacent to the codon.
A second channel through the large subunit provides an exit path for the
teraction are shown. The strong
mRNA clearly distinguishes b
A-site and P-site codons. The clo
of the 30 ends of the A-site and
newly synthesized polypeptide chain (Fig. 15-21). As with the mRNA chan-
can be seen in the lower imag
nel, the size of the peptide exit channel limits the conformation of the grow- M.M. et al. 2001. Science 292:
ing polypeptide chain. In this case, a polypeptide can form an a helix within Image prepared with Mol-Scrip
• The mRNA enters and exits the decoding center through two narrow channels in the small
the channel, but other secondary structures (such as b sheets) and tertiary
interactions can form only after the polypeptide exits the large ribosomal
and Raster3D.

subunit. subunit. For this reason, the final 3D structure of a newly synthesized pro-
tein is not attained until after it is released from the ribosome.
• The entry channel is only wide enough for unpaired RNA to pass through.
Translation 527 P A
E

enter in the small sub-

coupled to the amino FIGURE 15-21 The polyp
the large subunit. The tunnel. In this image, the 50
cut in half to reveal the polyp
adjacent to the small tunnel. The rRNA is white; th
proteins are yellow. The three b
are colored as follows: E-site (br
( purple), and A-site (green). T
gold parts of the rRNA adjac
A-site tRNA are components of
and Growing transferase center. (Courtesy o

©2002 Macmillan
Schmeing and Thomas Steit
with permission, from Schme
al. 2002. Nat. Struct. Biol. 9: 2
exit tunnel
Macmillan.)
rase center are buried
ded through the decod-
• A second
lypeptide chain must channel through the large subunit provides an exit path for the newly synthesized
polypeptide
o these polymers enter chain.
answer is provided by
els” in and out of the
3' 3'
(in the case of mRNA) and exit the ribosome? The answer is provided by
the structure of the ribosome, which reveals “tunnels” in and out of the
ribosome.
Ribosomal RNAs are both Structural and Catalytic
The mRNA enters and exits the decoding center through two narrow
channels in the small subunit. The entry channel is only wide enough for
3' 3'

F I G U R E 15-20 The interaction be-

Determinants of the Ribosome

unpaired RNA to pass through. This feature ensures that the mRNA is in a
single-stranded form as it enters the decoding center by removing any intra-
molecular base-pairing interactions that may have formed in the mRNA. In
tween the A-site and P-site tRNAs and
the mRNA within the ribosome. Two
views of the structure of the mRNA and
tRNAs are shown as they are found in the ri-
between the two channels is a region that is accessible to tRNAs and where
bosome. For clarity, the ribosome is not
adjacent codons can bind to the aminoacyl-tRNA and peptidyl-tRNA in the
shown. The E-, P-, and A-site tRNAs are
A- and P-sites, respectively. Interestingly, there is a pronounced kink in the
• Peptidyl transferase center of the large subunit is composed almost entirely of RNA.
mRNA between the two codons that facilitates maintenance of the correct
shown in yellow, red, and green, respective-
ly, and the mRNA is shown in blue. Only the
reading frame (Fig. 15-20). This kink places the vacant A-site codon in a dis- bases involved in the codon–anticodon in-

• The anticodon loops of the charged tRNAs and the codons of the mRNA contact the 16S rRNA of the
tinctive position that ensures that the incoming aminoacyl-tRNA does not
have access to bases immediately adjacent to the codon.
teraction are shown. The strong kink in the
mRNA clearly distinguishes between the

small subunit.
A second channel through the large subunit provides an exit path for the A-site and P-site codons. The close proximity
of the 30 ends of the A-site and P-site tRNAs
newly synthesized polypeptide chain (Fig. 15-21). As with the mRNA chan-
can be seen in the lower image. (Yusupov
nel, the size of the peptide exit channel limits the conformation of the grow-
• Most ribosomal proteins are on the periphery of the ribosome, not in its interior.
M.M. et al. 2001. Science 292: 883– 896.)
ing polypeptide chain. In this case, a polypeptide can form an a helix within Image prepared with Mol-Script, BobScript,
the channel, but other secondary structures (such as b sheets) and tertiary and Raster3D.
interactions can form only after the polypeptide exits the large ribosomal
• Function of those reaching core seems to be to stabilize the rRNAs by shielding the negative charges
subunit. For this reason, the final 3D structure of a newly synthesized pro- of
Translation 525

their sugar–phosphate backbones.

tein is not attained until after it is released from the ribosome.

P A
E

FIGURE 15-21 The polypeptide exit

tunnel. In this image, the 50S subunit is
cut in half to reveal the polypeptide exit
tunnel. The rRNA is white; the ribosomal
proteins are yellow. The three bound tRNAs
are colored as follows: E-site (brown), P-site
( purple), and A-site (green). The red and
gold parts of the rRNA adjacent to the
A-site tRNA are components of the peptidyl
transferase center. (Courtesy of T. Martin
©2002 Macmillan

Schmeing and Thomas Steitz; adapted,

with permission, from Schmeing T.M. et
al. 2002. Nat. Struct. Biol. 9: 225–230. #
exit tunnel
Macmillan.)
 AU
P A
5' 3'

Initiation Of Translation For translation to be successfully initiated, three events must oc

15-22): the ribosome must be recruited to the mRNA; a charge
must be placed into the P-site of the ribosome; and the ribosome mus
fMet
cisely positioned over the start
528
codon. The correct positioning of
Chapter 15
some over the start codon is critical because this establishes the
• For translation to be successfully initiated,
E P A
three events must occur:
frame for the translation of the mRNA. Even a 1-base shift Nowin thatthe
we
1. Recruitment of ribosome to mRNA.
fMet

U A CG
of the ribosome would result in the
large synthesis of
initiator a completely
tion process, u
the
U tRNA of translation. O
A polypeptide (see the discussion of mRNA aboveC and in Chapter 16).
2. Precise placement of ribosome
5' over the start codon.
3'
E P A UA
similar structures of prokaryotic and eukaryotic mRNAs
of translation: in
result
tion in d
of the grow

3. Placement of a charged tRNA into the P-site of the different ribosome. means of accomplishing these events. We start by addres
initiation events in prokaryotes and mRNA
As we shall
prokaryotes and
see
F I G U R E 15-22 An overview of the small then discuss the differences
events. We shao
• mRNAs are initially recruited to the small subunit by baseinpairing
events of translation initiation.
tocells.
eukaryotic rRNA. both kinds of ce
DNA and RNA
auxiliary factors
• For ideally positioned RBSs, the small subunit is positioned on the mRNA
Prokaryotic mRNAs Are Initially Recruited to the Small Subunit fMet
are required for

such that the start codon will be in the P-site. by Base Pairing to rRNA
UAC INITIATION
The assembly of the ribosome on an mRNA AU
P A
G
occurs one subunit at a tiO
ribosome- 16S start 30S small subunit associates with 5' the mRNA first. As 3' described during
For translation
binding site rRNA codon subunit cussion of mRNA structure (see Fig. 15-2), in prokaryotes, thetheassoc
15-22): ribo
P A the small subunit with the mRNA is mediated by base-pairing intei
must be placed
between the RBS and the 16S rRNA (Fig. 15-23). Forcisely
fMet
positione
ideally po
5' AUG 3' some over the s
RBSs, the small subunit is positioned E on
P the
A mRNA suchframe thatforthethesta
tra
will be in the P-site when the large subunit U A CG joins the complex. The la
of the ribosome
AU
unit joins its partner only at the very end of the initiationpolypeptide process,
similar
(see
ju
structure
5' 3'
16S rRNA the formation of the first peptide bond. Thus, many of the key events
different means
3' A U A G 5'
UCCUCCACU lation initiation occur in theF I Gabsence
U R E 15-22ofAnthe full ofribosome.
overview the initiation events
events of translation initiation. in eukaryotic ce
5' N N N A
GGA GN N N N N N
N N A U G N N 3'
mRNA
A Specialized tRNA Charged with a Modified Methionine Binds
Prokaryotic mR
by Base Pairing
Directly to the Prokaryotic Small Subunit
FIGURE 15-23 The 16S rRNA inter- The assembly of
acts with the RBS to position the AUG in Translation initiation is the only
ribosome-time
16S a tRNA
start binds
30S to the P-site
small with
subunit as
 both
DNA

Initiation Of Translation
auxi
are r
fMet

U A CG INI
• A specialized, initiator tRNA, charged with a modified Methionine AU
P A
binds directly to the small subunit at the P-site (only time a tRNA 5' 3'
For
directly binds the P-site). TA B L E 16-1 The Genetic Code 15-2
second position
mus
• The modification is the addition of a formyl group by a separate
U C A G

UUU UCU fMet UAU UGU U

cise
Phe Tyr Cys
som
enzyme (Met-tRNA transformylase). U
UUC
UUA
Leu
UCC

E
UCA
Ser
P
UAC

A
UAA* stop
UGC
UGA* stop
C
A
fram
UUG UCG UAG* stop UGG Trp G
of th
⇒ The charged initiator tRNA is referred to as fMet-tRNAifMet CUU
CUC
CCU
CCC
U A CG
A U CAU His
CAC
CGU
CGC
U
C
poly

third position (3' end)

first position (5' end)
C
5'
CUA
Leu
CCA
Pro
CAA CGA
Arg
3' A simi
• An enzyme known as a deformylase subsequently removes the CUG CCG CAG
Gln
CGG G
diffe
formyl group. A
AUU
AUC Ile
events of ACA
15-22
F I G U R E ACU
ACC
translation
Thr
AAU
An
AAC
overview
Asn
AGU
AGC
[Link]
of U
Ser the
C
initi
in eu
AUA AAA A
Lys Arg

• In fact, many mature prokaryotic proteins do not even start with a AUG† Met

GUU
ACG

GCU
AAG

GAU
AGG

GGU
G

methionine: G
GUC
Val
GCC
Ala
GAC
Asp
GGC
Gly
C Prok
GUA
GUG
GCA
GCG
GAA
GAG
Glu
GGA
GGG
A
G
by B
• aminopeptidases often remove the amino-terminal methionine as * Chain-terminating or “nonsense” codons.
† Also used in bacteria to specify the initiator formyl-Met-tRNAfMet. The
well as one or two additional amino acids. CH3
ribosome- 16S start
CH3
30S sma
binding site rRNA codon subunit cuss
S S
P A the s
3'
O HCH H
2 CH3
3'
CHO2 H H CH3
betw
A O C C C 5' CH2 AUG A O C C C 3' CH2
C H CH2 CH3
O C
H
C
CH2 CH3
RBS
C NH3 H NH3 H

5' G
A
C H acceptor
N
leucine
C COOH H 5'
CG N
A
U C
leucine
COOH
will
C
G
G stem
C
C G
C G unit
A U H G C H
A C
ψ
C T C stem
16S rRNA
A U
G C
the f
G methionine 3' A N-formyl
G 5'
methionine (fMet)
D stem G C G U U C C U C C A C U AG CU G C C C U G A latio
D AA U C C C C CC A D AA U
D GGCGG A TψC D GGUGG A
D Gm G U RC E 15-24
G G GFGI G loop N A G GGAm Gand
Methionine N N N-formyl
N methionine.
AC GG GT ψC
 Initiation Of Translation: Initiation Factors 530 Chapter 15

a
• Three Initiation Factors direct the assembly of an Initiation Complex 50S bin
that contains mRNA and the initiator tRNA: E P A
E P A ca
+ ab
• IF1 prevents tRNAs from binding to the A-site. 30S th
• IF2 is a GTPase that interacts with three key components of the th
an
initiation machinery: the small subunit, IF1, and the charged IF3 th
initiator tRNA. It facilitates the association of initiator tRNA with fM
the small subunit and prevents other charged tRNAs from b
cre
associating with the small subunit. ba
• IF3 binds to the small subunit and blocks it from reassociating 3
alt
th
with a large subunit (IF3 becomes associated with the small E P A
IF
subunit at the end of a previous round of translation when it IF2
+ IF1 + GTP sit
helps to dissociate the 70S ribosome into its subunits). GT
rib
c GTP
2
3 1 as
E of
• Each of the initiation factors bind at, or near, one of the three tRNA- P A
fM
binding sites on the small subunit. fMet
co
fMet-tRNA mRNA me
fMet
d GTP Eu
2
3 1
E In
 P A
IF2
IF2
+ IF1 + GTP sit

Initiation Of Translation: Initiation Factors c 2

GTP
GT
rib
3 1 as
E P A of
fM
• With the three IFs bound, the small subunit is prepared to bind to the fMet
co
mRNA and the initiator tRNA. fMet-tRNA mRNA me

• Binding of mRNA is mediated by RBS-16S base pairing.

fMet
d GTP Eu
2
3 1
• Binding of fMet-tRNAifMet is
facilitated by its interactions with IF2-GTP, E
P A
Ini
ma
30S initation
and base pairing with the start codon. complex bo
un
3 Ne
the
• The last step of initiation involves the association of the large subunit. eu

• When the start codon and the initiator tRNA base-pair, the small subunit e fMet tR
GTP
undergoes a change in conformation, resulting in the release of IF3. E 2 A
1
alo
the
• IF2 acts as an initial docking site of the large subunit, and this interaction wh
P

15
subsequently stimulates the GTPase activity of IF2-GTP. 2 + GDP + P i AU
1
f tha
• IF2 bound to GDP has reduced affinity for the ribosome and the initiator fMet
of
tRNA, leading to the release of IF2-GDP as well as IF1 from the ribosome. E A tio
P
70S initation co
complex tei
ini
FIGURE 15-25 A summary of transla- tio
tion initiation in prokaryotes. sm
 R E 15-17 Two views of the ribosome. The 50S subunit is above the 30S subunit in both charged
The cavity between the 50S and 30S subunits in the right-hand image represents the site of we discu
ssociation (see Fig. 15-19b). The RNA component of the 50S subunit is shown in gray; the Acids to t

Translation Elongation
n component of the 50S subunit is shown in purple; the RNA component of the 30S
t is shown in light blue; the protein component of the 30S subunit is shown in dark blue.
ov M.M. et al. 2001. Science 292: 883–896.) Images prepared with MolScript, BobScript,
group of
manner (
chain. Th
ster3D. ken duri
between
as the gr
• Three
bosome has vastly keyour
increased events must of
understanding occur for the
the workings correct addition of each amino acid :
of this
polypeptide chain
H O
H H
attached
cular machine (Fig. 15-17). Perhaps the most important outcome of To cat
[Link],
studies is the finding rRNAs are themuch
correct
more thanaminoacyl-tRNA
structural compo- is loaded into the A-site of the
N

H
C

R
C
N C R H 2N C R
brought i
ing allow
s of the ribosome. Rather, ribosome, dictated byforthe A-site
func-codon.
H C O C O
they are directly responsible the key O O attack the
of the ribosome. The most obvious example of this is the demonstration
he peptidyl transferase2. center
Second, a peptide
is composed almost bond
entirely is formed
of RNA, as between the aminoacyl-tRNA in
to the pep
of a new
ussed in detail later. RNAthe alsoA-site
plays a and
centraltherolepeptide chainof that is attached to the peptidyl-
in the function the relea
There are
mall subunit of the ribosome. tRNA in the P-site.
The anticodon loops of the charged tRNAs
this mech
he codons of the mRNA contact the 16S rRNA, not the ribosomal pro- of the pr
of the small subunit. 3. As the growing polypeptide is transfered to the amino acid moiety peptidyl-tRNA aminoacyl-tRNA
growing
of the charged tRNA in the A-site, the resulting peptidyl-tRNA in
further indication of the importance of RNA in the structure and func-
of the ribosome is that most ribosomal proteins are on the periphery of
aminoacy
is called
bosome, not in its interiorthe(Fig. A-site and
15-17; see also its associated
Structural codon must be translocated to the P-
Tutorial 15-1). H O
Interes
ous hydr
core functional domains of site.
the ribosome (the peptidyl transferase cen- N C C
N
R

C
O

C N
H

C R formation
nd the decoding center) are composed either entirely or mostly from H R
ing polyp
H H H C O
. Portions of some ribosomal proteins do reach into the core of the sub- O
the tRNA
, where their function seems to be to stabilize the tightly packed rRNAs OH
tRNA. Th
hielding the negative charges of their sugar –phosphate backbones. Thus, the
of ATP th
ed, it is likely that the contemporary ribosome evolved from a primitive
in-synthesizing machine that was composed entirely of RNA and that E P A
bosomal proteins were added to enhance the function of this primor- Ribosom
RNA machine. mRNA Determin
5' 3'
FIGURE 15-16 The peptidyl transfer- Although
ase reaction. years ago
Ribosome Has Three Binding Sites for tRNA FIGURE 15-18 The ribosome has
three tRNA-binding sites. The schematic il-
erform the peptidyl transferase reaction, the ribosome must be able to lustration of the ribosome shows the three
 in the A-site and the peptide chain that is attached to the peptidyl-tRNA in the
fMet
P-site. This peptidyl transferase reaction, as we have seen, results in the trans-

Translation Elongation
fer of the growing polypeptide from the tRNA in the P-site to the amino acid
E P A
moiety of the charged tRNA in the A-site. Third, the resulting peptidyl-tRNA
in the A-site and its associated codon must be translocated to the P-site so that U A CGA C
AU A
the ribosome is poised for another cycle of codon recognition and peptide-
bond formation. As with the original positioning of the mRNA, this shift 5' 3'
must occur precisely to maintain the correct reading frame of the message. Thr
Two auxiliary proteins known as elongation factors control these events. aminoacyl-tRNA
• Three key events must occur for the correct addition of each amino acid :
Both of these factors use the energy of GTP binding and hydrolysis to enhance binding to A site
the rate and accuracy of ribosome function.
1. First, the correct aminoacyl-tRNA is loaded into the A-site of the
U
Unlike the initiation of translation, the mechanism of elongation is highly UG

conserved between prokaryotic and eukaryotic cells. We limit our discus-

ribosome, dictated by the A-site codon. sion to translation elongation in prokaryotes, which is understood in the
greatest detail, but the events that occur in eukaryotic cells are similar to
fMet Thr

2. Second, a peptide bond is formed between the aminoacyl-tRNA in

those in prokaryotes, both in the factors involved and in their mechanism
of action.
E P A
U
U A CGA CG U
the A-site and the peptide chain that is attached to the peptidyl- AU A

tRNA in the P-site. Aminoacyl-tRNAs Are Delivered to the A-Site by

5' 3'

Elongation Factor EF-Tu peptide bond

3. As the growing polypeptide isAminoacyl-tRNAs
transfereddoto not the
bind toamino acid
the ribosome moiety
on their own. Instead, they
formation

of the charged tRNA in the A-site, the resulting peptidyl-tRNA in

are “escorted” to the ribosome by the elongation factor EF-Tu (Fig. 15-30).
Once a tRNA is aminocylated, EF-Tu binds to the tRNA’s 3 end, masking 0 peptide bond

the A-site and its associated codon

the coupledmust be This
amino acid. translocated
interaction preventsto
thethe
boundP-aminoacyl-
tRNA from participating in peptide-bond formation until it is released
fMet
Thr

site. from EF-Tu. E P A

Like the initiation factor IF2, the elongation factor EF-Tu binds and U
hydrolyzes GTP, and the type of guanine nucleotide bound governs its func- U A CGA CG U
AU A
tion. EF-Tu can only bind to an aminoacyl-tRNA when it is associated with
5' 3'
GTP. EF-Tu bound to GDP, or lacking any bound nucleotide, shows little
affinity for aminoacyl-tRNAs. Thus, when EF-Tu hydrolyzes its bound
GTP, any associated aminoacyl-tRNA is released. On its own, EF-Tu bound translocation
to an aminoacyl-tRNA does not hydrolyze GTP at a significant rate. Instead,
the EF-Tu GTPase is activated when it associates with the same domain on
the large subunit of the ribosome that activates the IF2 GTPase when the fMet
Thr
large subunit joins the initiation complex. This domain is known as the
E
factor-binding center. EF-Tu only interacts with the factor-binding center P A
after the tRNA enters the A-site and a correct codon – anticodon match is
made. At this point, EF-Tu hydrolyzes its bound GTP and is released from GU
CU A
UA GAC
the ribosome (Fig. 15-30). As we shall discuss later, control of GTP hydrol- AU
5' 3'
ysis by EF-Tu is critical to the specificity of translation.
FIGURE 15-29 Summary of the steps
 538 Chapter 15

Translation Elongation: Elongation Factors polypeptide chain translatio

specificit
factor-binding center
incorrect
One m
involves t
• Aminoacyl-tRNAs are delivered to the A-Site by Elongation Factor EF-Tu. E P A within the
the minor
the first tw
• Once a tRNA is aminocylated, EF-Tu binds to the tRNA’s 3’ end, masking 5' 3'
6, Fig. 6-1
A:U base
the coupled amino acid. EF-Tu–GTP idues in t
GTP and recog
• EF-Tu can only bind to an aminoacyl-tRNA when it is associated with GTP. aa
or mismat
bases, res
result of th
• EF-Tu’s GTPase activity is activated when it associates with the same aminoacyl-tRNA
rate of dis
A seco
domain on the large subunit of the ribosome that activates the IF2 GTPase. ing involv
GTP above, rel
• This domain is known as the factor-binding center. E P
aa
A
highly sen
match in
reducing
• EF-Tu only interacts with the factor-binding center after the tRNA enters leads to a
is an exam
the A-site and a correct codon–anticodon match is made. 5' 3'
to ensure
both case
EF-Tu–GDP
• At this point, EF-Tu hydrolyzes its bound GTP and is released from the GDP
+ Pi
enhances
this step
ribosome. the hydro
A third
ing that oc
aa
duced int
the site of
E P A tidyl trans
center of t
During ac
5' 3'
70 Å. Inco
ing accom
FIGURE 15-30 EF-Tu escorts amino- strain on
acyl-tRNA to the A-site of the ribosome. anticodon
 Translation Elongation:
Selecting against incorrect codon–anticodon pairings
a b cor
3' 5'
The Structure
correct anticodon

• At least three different mechanisms contribute to this specificity: pairing

The Major Groove loop of
Is Rich in Chemical Information
tRNA
The edges of each base pair are exposed in the major and minor grooves, cre-
A C G
ating a pattern of hydrogen-bond donors and acceptors and of hydrophobic
mRNA

• First involves two adjacent adenine residues in the 16S rRNA component
groups (allowing for van der Waals interactions) that identifies the base pair E
(see Fig. 4-10). 5' U G C
The edge of an A:T base pair displays the3'following chemical
groups in the following order in the major groove: a hydrogen-bond acceptor
A A
located within the A-site of the small subunit.
(the N7 of adenine),
16S rRNAa hydrogen-bond donor (the exocyclic amino group
on C6 of adenine), a hydrogen-bond acceptor (the carbonyl group on C4 of 5'
thymine), and a bulky hydrophobic surface (the methyl group on C5 of
thymine). Similarly, the edge of a G:C base pair displays the following GTP hydrolysis
3'
groups in the major groove: a hydrogen-bond acceptor (at N7 of guanine), EF-Tu relea

• These bases form hydrogen bonds with the minor groove of each correct a hydrogen-bond acceptor (the carbonyl on C65'of guanine), a hydrogen-bond
donor (the exocyclic amino group on C4 of cytosine), and a small nonpolar
hydrogen (the hydrogen at C5 of cytosine).

base pair formed between the anticodon and the first two bases of the Thus, there incorrect
are characteristic patterns of hydrogen bonding and of over-
pairing
all shape that are exposed in the major groove that distinguish an A:T base
G C
pair from a G:C base pair, and, for that matter, A:T from T:A, and G:C from

codon in the A-site. U G C

G
C:G. We can think of these features as a code in which A represents a
hydrogen-bond acceptor, D a hydrogen-bond donor, M a methyl group,
and H a nonpolar hydrogen. In such a code, ADAM in the major groove sig-
5' 3'
E
nifies an A:T base pair, and AADH stands for a G:C base pair. Likewise,

• Recall that the hydrogen-bonding properties of a G:C and A:U base pair are
MADA stands for a T:A base pair, and HDAA is characteristic of a C:G
A A
base pair. In all cases, this code of chemical groups in the major groove
5'
specifies the identity of the base pair. These patterns are important because

very similar in the minor groove. c

major groove major groove inco

D D

• ⇒ the adjacent adenine residues recognize either as correct. A

N
A

O H
H

N
4 5
H

E
H
P
aa H
A
H

5 4
H

N H
A

O
A
N
7 7
6
H N C C N H
6
E

• Mismatched bases form a minor groove that are significantly different ⇒

G N 2 2 N G
N 2
N N N
3 2 3
N O O N
N 5' H H
3' N
A A A A

significantly reduced affinity. 5'

H H
D
accommodation D
minor groove minor groove no GTP hydro
aa
EF-Tu•tRNA relea

major groove major groove

D A M M A D
H aa H
A O CH3 CH3 O A
N H H N
N
E
7
P A 4 5 5 4E P A N
7
N T T N
A
6
N H
2 2
H N
6
A
E
N 2
N N 2
N
3 3
N O O N
H H FIGURE 4-10 Chem
5' 3' 5' 3' posed in the major and
correct
A Hbase A incorrect
A H base
A
along the 5' edges of the b
pairing pairing ters in red identify hydroge
(A), hydrogen-bond don
minor groove minor groove hydrogens (H), and methy
FIGURE 15-31 Three mechanisms to ensure correct pairing between the tRNA and the
 a b correct pairing
3' 5'
polypeptide chain

Selecting against incorrect codon–anticodon correct anticodon factor-binding

pairing loop of center
tRNA
GTP

pairings
A C G aa
mRNA
E P A
5' U G C 3'

16S rRNA A A 5' 3'

EF-Tu-GDP
GTP hydrolysis and
• Second involves the GTPase activity of EF-Tu. 3'
5'
EF-Tu released GDP
+ Pi

• Release of EF-Tu from the tRNA requires GTP hydrolysis,

incorrect which is highly
pairing
sensitive to correct codon – anticodon base pairing. G C
G aa

• Even a single mismatch alters the position of EF-Tu 5' U G C 3'

E P A

A A
• ⇒ reducing its ability to interact with the factor-binding center 5' 3'

• ⇒ dramatic reduction in EF-Tu GTPase activity

c
incorrect pairing
factor-binding
center not
contacted
• Example of kinetic proofreading: formation of correct base-pairing
E P A
aa GT
P

interactions dramatically enhances the rate of a critical biochemical step. E P A aa

5' 3'
5' 3'
accommodation
GTP
no GTP hydrolysis
aa
EF-Tu•tRNA released aa

aa

E P A E P A
E P A

5' 3' 5' 3'

correct base incorrect base
5' 3'
pairing pairing
 incorrect
pairing
G C
Translation Elongation: G
U G C
Selecting against incorrect codon–anticodon pairings
5' 3'

A A

• Third mechanism is a form of proofreading that occurs after EF-Tu is

released. E P
aa
A

• When the charged tRNA is first introduced into the A-site, its 3’ end is
distant from the site of peptide-bond formation. 5' 3'

accommodation

• The tRNA must rotate into the peptidyl transferase center in a process aa

called accommodation.
• Accommodation is thought to a strain on the codon–anticodon aa

interaction and that only a correctly paired anticodon can sustain this E P A E P A

strain.
5' 3' 5' 3'
correct base incorrect base
• Incorrectly paired tRNAs will dissociate during this step. pairing pairing

FIGURE 15-31 Three mechanisms to ensure correct pairing between the tRN
mRNA. (a) Additional hydrogen bonds are formed between two adenine residues
rRNA and the minor groove of the anticodon–codon pair only when the first two base
ticodon– codon pair form correct Watson–Crick base pairs. (b) Correct codon–antic
pairing facilitates EF-Tu bound to the aminoacyl-tRNA to interact with the factor-bindin
ducing GTP hydrolysis and EF-Tu release. (c) Only correctly base-paired aminoacyl-tRN
associated with the ribosome as they rotate into the correct position for peptide-bond
This rotation is referred to as tRNA accommodation.
 Peptidyle Transferase Reaction
• Peptidyle transferase reaction is catalyzed by 23S rRNA of the large subunit.
• Exact mechanism remains to be determined, but some answers have emerged:
• First, base pairing between the 23S rRNA and the CCA ends of the tRNAs in the A- and P-sites
positions the amino group of the aminoacyl-tRNA to attack the carbonyl group of the growing
polypeptide attached to the peptidyl-tRNA.
• Also likely to stabilize the aminoacyl-tRNA after accommodation.
• This type of catalytic mechanism is called entropic catalysis ⇒ enzyme works by bringing the
substrates together in a manner that stimulates catalysis.
 Peptidyle Transferase Reaction
• It is likely that other elements of the rRNA contribute to catalysis:
• Alterations that eliminate the 2’-OH of a highly conserved residue in the 23S rRNA reduce the rates
of catalysis by at least 10-fold.
• Mutations that remove the 2’-OH of the Adenine residue at the 3’ end of the P-site tRNA result in a
106-fold reduction in catalysis rates.
• It has been proposed that the 2’-OH of the P-site tRNA may act as part of a proton
Translation 541 shuttle.
polypeptide chain
H O H O
H H H H R O H H
N C C N C C
N C N C R N C C N C
H R H R
O C C O H H C O
H H
O O O OH OH OH O OH

3' 2' 3' 2' 3' 2' 3' 2'

H2C O Ade H2C O Ade H2C O Ade H2 C O Ade

O O O O

C C C C C C C C

peptidyl-tRNA aminoacyl-tRNA peptidyl-tRNA aminoacyl-tRNA

FIGURE 15-33 Proposed role for the 20 -OH of the P-site tRNA in peptide-bond formation.
The 20 -OH of the final “A” in the peptidyl-tRNA is critical for peptide-bond formation. Based on this
Peptide-Bond Formation Initiates Translocation in the Large Subunit
• The P-site tRNA must move to the E-site and the A-site tRNA must move to the P-site.
• At the same time, the mRNA must move by three nucleotides to expose the next codon.
• Collectively known as Translocation.
Peptide-Bond Formation Initiates Translocation in the Large Subunit
• The initial steps of translocation are coupled to the peptidyl transferase reaction.
• Once the growing peptide chain has been transferred, the A- and P-site tRNAs have a preference to
occupy new positions in the large subunit.
• In contrast, at this time, the anticodons of these tRNAs remain in their initial location in the small
subunit bound to the mRNA.
• ⇒ translocation is initiated in the large subunit before the small subunit.
• This change is associated with a counterclockwise rotation of the small subunit relative to the large
subunit, facilitating interaction of the tRNAs with distinct tRNA-binding sites.
542 Chapter 15

b a
FIGURE 15-34 EF-G stimulation of
hybrid state classic state
translocation requires GTP hydrolysis.
factor- aa
aa binding
P center P
E A small E A
subunit
rotation
5' 3' 5' 3'

GTP

EF-G–GTP
The initial steps of translocation are coupled to the peptidyl transferase
reaction (Fig. 15-34). Once the growing peptide chain has been transferred
 b a
hybrid state

EF-G Drives Translocation by Stabilizing E

aa
P
A
factor-
binding
center
small

Intermediates in Translocation
subunit
rotation
5' 3'

GTP

EF-G–GTP
The
reactio
to the
• Completion of translocation requires the action of a second elongation factor c new po
to the
called EF-G. aa
GTP large s
the gro
P subuni
• Initial binding of EF-G to the ribosome occurs when associated with GTP. E A
their in
locatio
5' 3' tRNAs
• After the peptidyl transferase reaction, EF-G – GTP binds to and stabilizes the new lo
fer pos
ribosome in the rotated, hybrid state d
P counte
facilita

• When EF-G–GTP binds, it contacts the factor-binding center ⇒ stimulates GTP GDP
differe

hydrolysis.
aa
EF-G D
PA in Tran
The co
5' 3' factor c
ciated
GDP
and sta
EF-G –
which
GTP
(Fig. 15
though
e
“gates”
the rib
format
aa
petes w
E P A Becaus
the P-s
stabiliz
5' 3' EF-G –
left the
 b
hybrid state

EF-G Drives Translocation by Stabilizing E

aa
P
A
factor-
binding
center
sm

Intermediates in Translocation
sub
rota
5' 3'

GTP

EF-G–GTP
rea
• GTP hydrolysis changes the conformation of EF-G with two consequences : to
c new

• First, interactions between EF-G–GDP and the ribosome are thought to “unlock” the GTP
to
lar

ribosome, by opening “gates” that separate the A-, P-, and E-sites. aa
P
A
the
sub
E the
• Second, the changed EF-G – GDP conformation binds to the A-site of the decoding 5' 3'
loc
tRN
center. new
fer
P cou
• This interaction competes with the tRNA for binding to the A-site of the decoding d
fac
dif
center. aa
GDP
EF

• Because the ribosome is unlocked, the formerly A-site tRNA can move into the P-site. PA in
Th

• Like dominoes, movement of the A-site tRNA into the P-site forces the P-site tRNA 5'
GDP
3' fac
cia

into the E-site. an

EF
wh
• Base pairing between the tRNAs and the mRNA causes the mRNA to move by 3 bp. (Fi
tho
e
“ga
the
for
aa
pe
E P A Be
the
sta
5' 3' EF
lef
 b
hybrid state

EF-G Drives Translocation by Stabilizing E

aa
P
A
factor-
binding
center
sm

Intermediates in Translocation
sub
rota
5' 3'

GTP

EF-G–GTP
rea
• Completion of translocation is accompanied by a clockwise rotation of the small to
c
subunit back to its starting position.
new
to
GTP lar

• The resulting ribosome structure has dramatically reduced affinity for EF-G–GDP. aa
P
A
the
sub
E the
• Release of EF-G results in the return of the ribosome to a “locked” state. 5' 3'
loc
tRN
new
fer
P cou
d
fac
• EF-Tu–GDP and EF-G–GDP must exchange GDP for GTP before participating in a new dif

round of elongation. aa
GDP
EF
PA in
• A cycle of peptide-bond formation consumes two molecules of GTP and one Th

molecule of ATP. 5'

GDP
3' fac
cia
an
• One molecule of ATP is consumed in charging the tRNA, one GTP for EF-Tu, and one EF
wh
GTP for EF-G. (Fi
tho
e
“ga
the
for
aa
pe
E P A Be
the
sta
5' 3' EF
lef
 Things remaining

• Chopping-off of the polypeptide chain from tRNA.

• Disassembly of the ribosomal subunits for next round of translation.
 Translation Termination
• When one of the three stop codons enters the A-site, proteins called release factors (RFs) associates
with it and terminates translation.
• There are two classes of release factors: Class I and Class II
• Class I release factors recognize the stop codons and trigger hydrolysis of the peptide chain from the
tRNA in the P-site.
• Prokaryotes have two Class I release factors called RF1 and RF2:
• RF1 recognizes the stop codon UAG, and RF2 recognizes the stop codon UGA. The third stop codon,
UAA, is recognized by both RF1 and RF2.

• Class II release factors (RF3) stimulate the dissociation of the class I factors from the ribosome after
release of the polypeptide chain.
 Translation Termination Translation

F I G U R E 15-37 3D structures of RF1 bound to the ri-

• How do release factors recognize stop codons? bosome. (a) This view shows RF1 binding to the A-site of
the ribosome. (b) This structure shows that the peptide an-
ticodon is located very near the anticodon. (c) In this view,
• Protein–RNA interaction must mediate stop codon recognition. the structure of RF1 bound to the ribosome shows the GGQ
motif located close to the 30 end of the P-site tRNA and the
peptidyl transferase center. (Adapted, with permission,

• Experiments have found that a three-amino-acid sequence is critical for release factor
from Petry et al. 2005. Cell 123: 1255 –1266. # Elsevier.)

specificity.
• ⇒ Exchange of these three amino acids between RF1 and RF2 swaps their stop-codon
specificity.
• It is located very near the anticodon.
• ⇒ this sequence is called a peptide anticodon.
 Translation Termination
• How do release factors recognize hydrolyze the peptide chain from tRNA?
• A region of Class I RFs that stimulates polypeptide release has been identified:
• A conserved three-amino-acid sequence (glycine glycine glutamine, GGQ)
• The structure of RF1 bound to the ribosome confirms that the GGQ motif is located close to
the peptidyl transferase center.
• It remains unclear whether it is directly involved in the hydrolysis of the polypeptide, or
• if it induces a change in the peptidyl transferase center allowing the center itself to catalyze
hydrolysis.
 CCA

Translation Termination
GGQ factor-binding
site
aa

RFI
P
E
A SPF

• Once hydrolysis of the peptidyl-tRNA linkage is complete, Class-I RF 5' 3'

must be removed from the ribosome.

peptide hydrolysis

GDP

Anticodon
• Stimulated by the Class II release factor, RF3, a GTP-binding protein. RF-3-GDP

aa

• However, it is the GDP bound complex that enters ribosome first.

FIGURE 15-38 Comparison of the structures of RF1 to a tRNA. The tRNA (dark red) and RF1
(gray) are shown occupying the same space. (Redrawn, with permission, from Petry et al. 2005. Cell
123: 1255 –1266, Fig. 3E. # Elsevier.)
GDP

• RF3-GDP binding to the ribosome is Class I RF dependent.

E
A

A region of class I release factors that stimulates polypeptide release has

• Release of polypeptide ⇒ a change insequence

the(glycine
conformation of) thatthe ribosome,
also been identified. All class I factors share a conserved three-amino-acid 5' 3'
glycine glutamine, GGQ is essential for polypeptide GTP

release. Moreover, the structure of RF1 bound to the ribosome confirms that

⇒ stimulates RF3 to exchange its bound ([Link]

15-38c). Itfor a unclear
GTP,whether the GGQ motif is directly involved
the GGQ motif is located in close proximity to the peptidyl transferase center
remains
in the hydrolysis of the polypeptide from the peptidyl-tRNA or if it induces a GDP

⇒ changes conformation of ribosome,changedisplacing

in the peptidylthe Class
transferase I factor,
center that allows the center itself to cat-
alyze hydrolysis. Studies of the conserved bases found adjacent to the CCA GTP
RF-3-GTP

⇒ also allow RF3 to associate with theseveral

factor-binding center of hydrolysis.
the large
ends in the peptidyl transferase center (e.g., A2541 or A2602) indicate that
of these residues are required for peptide Indeed, these
P A

subunit, bases appear to play a more important role in peptide release than they do E
in peptide-bond formation. A likely explanation for this difference is that
only proximal RNA residues can position a small water molecule for hydrol-
⇒ stimulates the hydrolysis of GTP ysis, but residues at many sites in the ribosomes can help position the larger
tRNAs for catalysis.
5'

GDP
3'

Together, these studies have led to the hypothesis that class I release fac-
• In the absence of a bound Class I factor,
tors the resulting
functionally RF3-GDP
mimic a tRNA, having has that
a peptide anticodon a low
interacts RF-3-GDP

affinity for the ribosome and is released.

with the stop codon and a GGQ motif that reaches into the peptidyl transfer- P
ase center. Comparison of the structure of RF1 to a tRNA reveals how the pro-
tein functionally mimics a tRNA (Fig. 15-38). Just as the CCA 30 terminus and
the anticodon loop occupy extreme ends of each tRNA, the GGQ and the
peptide anticodon loop occupy extreme ends of RF1.
P A
E

GDP/GTP Exchange and GTP Hydrolysis Control the Function

of the Class II Release Factor 5' 3'
 5' 3'
ysis, but residues at many sites in the ribosomes can help position the larger
tRNAs for catalysis. GDP

Release of Ribosome from mRNA and tRNAs

Together, these studies have led to the hypothesis that class I release fac-
tors functionally mimic a tRNA, having a peptide anticodon that interacts
with the stop codon and a GGQ motif that reaches into the peptidyl transfer-
RF-3-GDP

P
ase center. Comparison of the structure of RF1 to a tRNA reveals how the pro- 548 Chapter 15
tein functionally mimics a tRNA (Fig. 15-38). Just as the CCA 30 terminus and
• To participate in a new round of polypeptide synthesis:
the anticodon loop occupy extreme ends of each tRNA, the GGQ and the of the r
peptide anticodon loop occupy extreme ends of RF1. bound
1. the tRNAs and the mRNA must be removed from the ribosome, E
P A
RF3 in
RF3 le
2. the ribosome mustGDP/GTP
dissociate intoanditsGTPlarge
Exchange and Control
Hydrolysis smallthe
subunits.
Function
5' 3'
that fa
of the Class II Release Factor mediat
• Collectively, these events are
Once the referred
class to has
I release factor as triggered
ribosome recycling.
the hydrolysis of the peptidyl- FIGURE 15-39 Polypeptide release is
from t
catalyzed by two release factors. The class factor-
tRNA linkage, it must be removed from the ribosome (Fig. 15-39). This
I release factor (shown here as RF1) re- teins i
step is stimulated by the class II release factor, RF3. RF3 is a GTP-binding
cognizes the stop codon and stimulates poly- GTP. In
protein but, unlike the other GTP-binding proteins involved in translation,
• A protein known as thethis
ribosome
factor has a recycling
higher affinityfactor
for GDP(RRF) cooperates
than GTP. Thus, free RF3with
is predom-
peptide release through a GGQ motif that is
localized to the peptidyl transferase center.
low aff

EF-G and IF3 to recycleinantly

ribosomes after polypeptide
in the GDP-bound form. RF3-GDP release
binds to the ribosome in a manner The class II E
P A
release factor (RF3) binds only
that depends on the presence of a class I release factor. After the class I after polypeptide release and drives the dis-
The Ri
release factor stimulates polypeptide release, a change in the conformation sociation of the class I release factor.
5' 3'
After t
RRF RRF binds in A site some i
E-sites
and th
dissoc
referre
PA In p
E
(RRF)
release
5' 3' mimic
GTP
that mi
EF-G–GTP of the u
this re
 RF3
that

Release of Ribosome from mRNA and tRNAs

med
from
facto
tein
GTP
low
• RRF binds to the empty A-site of the ribosome, where it mimics a tRNA. E
P A

The
• RRF also recruits EF-G – GTP to the ribosome. 5' 3'
Afte
som
• In events similar to translocation, the EF-G stimulates the release of the RRF RRF binds in A site
E-sit
and
uncharged tRNAs bound in the P- and E-sites. diss
refer
• Once the tRNAs are removed, EF-G–GDP and RRF are released from the E
PA
(RR
In

ribosome along with the mRNA. 5' 3'

relea
mim
GTP
that
• The final outcome of these events is a small subunit bound to IF3. EF-G–GTP of th
this
• IF3 may also participate in the release of the mRNA and is required to separate GTP
A-si
the
the two ribosomal subunits from each other. PA
and
tiati
E
requ
outc
5' 3' mRN
in a
R
bles
P
i
ribo
ciate
GDP this
way
E P A then
EF-G
the r
5' 3' recy
IF3
GDP som
and