IX.

GENE EXPRESSION: TRANSLATION

Dr. Riris Istighfari Jenie, M.Si., Apt

riris_jenie@ugm.ac.id
 Content:
•  Ribosom, kodon antikodon , tRNA, aminoasyl
tRNA sintetase
•  Proses : inisiasi, elongasi, dan terminasi
•  Perbedaan proses translasi pada prokariot dan
eukariot.
•  Pasca translasi : modifikasi asam amino.
•  Obat-obat dengan target penghambatan proses
translasi prokariot (antibiotik).
•  Teknik deteksi ekspresi gen
The Central Dogma

The central dogma states

that information in nucleic
acid can be perpetuated or
transferred, but the
transfer of information
form into protein is
irreversible.
From RNA to protein: translation
The genetic code

Three possible “reading frames”

THE ABC FOR THE DNA

Insertion (X)
or
Deletion (B)

THE AXB CFO RTH EDN A

THE ACF ORT HED NA

 Gene Expression and Regulation
Genes Can Be Expressed with Different Efficiencies at Different Times

and Environments

 Translation

•  Process of converting information stored in

nucleic acid sequences into proteins.
•  Sequences of mRNA (messenger RNA) are
translated into unique sequence of amino acids
in a polypeptide chain.
•  Takes place in the cytoplasm.
–  Exception are few proteins coded by mitochondrial
and chloroplastic DNA.
•  Performed on ribosomes.
Basic Principles of Transcription and
Translation
•  RNA is the bridge between genes and the
proteins for which they code
•  Transcription is the synthesis of RNA using
information in DNA
•  Transcription produces messenger RNA
(mRNA)
•  Translation is the synthesis of a polypeptide,
using information in the mRNA
•  Ribosomes are the sites of translation

© 2011 Pearson Education, Inc.

 •  In prokaryotes, translation of mRNA can begin
before transcription has finished
•  In a eukaryotic cell, the nuclear envelope
separates transcription from translation
•  Eukaryotic RNA transcripts are modified through
RNA processing to yield the finished mRNA

© 2011 Pearson Education, Inc.

 •  A primary transcript is the initial RNA
transcript from any gene prior to
processing
•  The central dogma is the concept that
cells are governed by a cellular chain of
command: DNA → RNA → protein

© 2011 Pearson Education, Inc.

 Materials?

1.  Ribosome
2.  Template: mRNA
3.  tRNAs (transfer RNAs)
4.  Accessory proteins
5.  Some energy (GTP hydrolysis)
 addition disrupts this high-energy covalent linkage, but immediately replaces it
with an identical linkage on the most recently added amino acid (Figure 6–59).

Ribosomes
In this way, each amino acid added carries with it the activation energy for the
addition of the next amino acid rather than the energy for its own addition—an
example of the “head growth” type of polymerization described in Figure 2–44.

•  TheRNA-protein
RNA Message Is Decoded complexes in Ribosomes (ribonucleoproteins)
MBoC6 m6.61/6.59
The synthesis of proteins is guided by information carried by mRNA molecules. To
•  maintain
Place of translation
the correct (protein
reading frame and to ensure synthesis)
accuracy (about 1 mistake every
10,000 amino acids), protein synthesis is performed in the ribosome, a complex

•  catalytic
Abundant
proteins) inmolecules,
cellsthe that ribosomal synthesize
machine made from more than 50 different proteins (the ribosomal
and several RNA RNAs (rRNAs). A typical large amounts of
protein
eukaryotic cell contains millions of ribosomes in its cytoplasm (Figure 6–60). The
large and small ribosome subunits are assembled at the nucleolus, where newly
transcribed and modified rRNAs associate with the ribosomal proteins that have
•  been
Structurally andafter
transported into the nucleus functionally
their synthesis in thesimilar among
cytoplasm. These two species (differ
ribosomal subunits are then exported to the cytoplasm, where they join together
to between prokaryotes and eukaryotes)
synthesize proteins.

Figure 6–60 Ribosomes in the cytoplasm

of a eukaryotic cell. This electron
micrograph shows a thin section of a small
region of cytoplasm. The ribosomes appear
as black dots (red arrows). Some are
free in the cytosol; others are attached to
membranes of the endoplasmic reticulum.
400 nm (Courtesy of Daniel S. Friend.)
 Ribosomes

•  Composed of small and large subunit

•  Subunits bind together for translation
•  Ribosomes self-assemble without additional
factors
 Ribosomes

•  Decoding and synthesis takes

place in the cavity between
subunits
•  Ribosomes move along mRNA
chain during translation
•  New peptide exits through the
tunnel in the large subunit
 Ribosomes
•  Contain rRNA molecules and
proteins
•  In prokaryotes
- Large subunit contains rRNA (5S
and 23S) and 32 proteins
- Small subunit contains 16S rRNA
and 21 proteins
•  In eukaryotes
- Large subunit contains rRNA (5S,
5.8S and 28S) and 49 proteins
- Small subunit contains 18S rRNA
and 33 proteins
 The RNA message is decoded in
FROM RNA TO PROTEIN ribosomes 341
= A complex catalytic machine made from ribosomal proteins and ribosomal RNAs
(rRNAs) 70S 80S

MW 2,500,000
MW 4,200,000
60S (large) subunit 40S (small) subunit
50S (large) subunit 30S (small) subunit

MW 1,600,000 MW 900,000 MW 2,800,000 MW 1,400,000

5S rRNA 23S rRNA 16S rRNA 5S rRNA 28S rRNA 5.8S rRNA 18S rRNA

120 120 160

nucleotides 2900 1540 nucleotides nucleotides 1900
nucleotides nucleotides nucleotides
4700
nucleotides

34 proteins 21 proteins ~49 proteins ~33 proteins

BACTERIAL RIBOSOME EUKARYOTIC RIBOSOME Fig 6-61

 Ribosomes
•  Cytosolic (free)
•  Bound to ER
•  Also located in mitochondria and chloroplasts of eukaryotic
cells
•  Free ribosomes
- Found in the cytosol
- May exist as a single ribosome or in groups known as
polysomes
- Occur in greater number than bound ribosomes in cells that
retain most of their manufactured protein in the cytosol
•  Bound ribosomes
- Bound to the exterior of the rough endoplasmic reticulum
- Occur in greater number than free ribosomes in cells that
secrete their manufactured proteins (e.g., pancreatic cells)
Composition of eukaryotic ribosomes
 RNA-binding sites in the ribosome
Each ribosome has 4 binding sites for RNA molecules:
•  a binding site for mRNA
•  three binding sites for tRNA

•  A-site: aminoacyl-tRNA
•  P-site: peptidyl-tRNA
•  E-site: exit

 (Eukaryotic) mRNA

•  Single stranded molecule of RNA that encodes

sequence of the polypeptide
•  Transcribed and processed in the nucleus and then
exported into cytoplasm
•  5’ cap - methylated base
–  Protects from nucleases
–  Binding sites for translation initiation
•  Middle is a coding sequence - codes for one protein
•  3’ end - poly-A tail
–  50-200 adenines added post-transcriptionally
–  Protects mRNA from degradation
–  Regulates stability of mRNA
 (The late 1950s)
The “coding problem”:
how is the information in a linear sequence
of nucleotides in RNA translated into the
linear sequence of a chemically quite
different set of units - the amino acids in
proteins?

From DNA to protein

 The Genetic Code
•  Generally, the correspondance between the
information stored in the language of nucleid acid and
protein
•  Defines how the message is translated (”a nucleid acid
–amino acid dictionary”)
•  More specifically, the correspondance between
triplets of nucleotides in the mRNA (read from 5’ to 3’)
and amino acids in protein (read from N-terminus to C-
terminus)

 Genetic Code:
(3 nonsense; 61 coding yet fewer tRNA)

start
The Genetic Code
- 3 nonsense; 61 coding yet fewer tRNA

-sequence of nucleotides in the mRNA

molecule is read in consecutive groups of
three (codon)
-4 × 4 × 4 = 64 possible combinations of
three nucleotides
Codon = group of three consecutive
nucleotides = triplet
Start codon (in green)
•  AUG
Stop codons (in orange)
•  UAA, UAG, UGA
Redundant (usually the 3rd nucleotide),
because 61 codons for 20 amino acids
 Interpretation of the Code
•  The meaning of a codon that represents an amino acid is
determined by the tRNA that corresponds to it
•  This requires base pairing between the codon in mRNA with the
anticodon of the tRNA within the ribosome
•  The meaning of the termination codons is determined directly by
the protein factors
•  Chemically similar amino acids are represented by related codons
to minimize the effect of mutations
•  Identical in almost all living organisms (differences in the code of
mitochondrial DNA)
4 Chapter 6: How Cells Read the Genome: From DNA to Protein
The genetic code
AGA UUA AGC
AGG UUG AGU
GCA CGA GGA CUA CCA UCA ACA GUA
GCC CGC GGC AUA CUC CCC UCC ACC GUC UAA
GCG CGG GAC AAC UGC GAA CAA GGG CAC AUC CUG AAA UUC CCG UCG ACG UAC GUG UAG
GCU CGU GAU AAU UGU GAG CAG GGU CAU AUU CUU AAG AUG UUU CCU UCU ACU UGG UAU GUU UGA

Ala Arg Asp Asn Cys Glu Gln Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val stop

A R D N C E Q G H I L K M F P S T W Y V

Initiation codon, Termination sites,

Start codon, stop codon
Methionine
mRNA Sequence
•  Codons areIs always
Decodedwritten
in Setswith
of Three
the Nucleotides
Figure 6–48 The genetic code. The
5’-terminal nucleotide to the left standard one-letter abbreviation for eac
•  Most amino acids are represented by more than one codon
ce an mRNA has been produced by transcription and processing, the informa- amino acid is presented below its three-
•  Used universally in all present-day organisms
n present in its nucleotide sequence is used to synthesize a protein. Transcrip- letter abbreviation (see Panel 3–1, pp. 1
113, for the full name of each amino aci
n is simple to understand as a means of information transfer:
MBoC6 m6.50/6.48 since DNA and and its structure). By convention, codon
A are chemically and structurally similar, the DNA can act as a direct template Figure 6-48
 Transfer RNAs (tRNAs)
•  Adaptor molecules that match amino acids to codons in
mRNA
•  Any cell contains different types of tRNA molecules
sufficient to incorporate all 20 amino acids into protein
•  Some tRNAs can recognise more than one codon
•  About 80 nucleotides in length
Structures of tRNAs
All tRNAs share a general
common structure that includes:

•  an acceptor stem
(to which the amino acid is
attached)

• an anticodon triplet loop
(pairs with mRNA codons)

Structures of tRNAs
 tRNAs are covalently modified before
they exit
FROM from
RNA TO the nucleus
PROTEIN

•  Eukaryotic tRNAs: synthesized by RNA O pol III H

O
H H
•  Bacterial and eukaryotic tRNAs: N synthesizedN as larger precursor
H N
tRNAs: H
CH3 H
N N O
–  Trimming: to produce the mature tRNA
N
H
N
CH3
–  tRNA splicing: someP tRNA precursors contain introns, P a cut-
and-paste mechanism, catalyzed
ribose by proteins ribose

•  Correctly folded in its cloverleaf configuration

two methyl groups added to G two hydrogens added t
(N,N-dimethyl G) (dihydro U)
–  Affect the conformation and base-pairing of anticodon
–  Facilitate the recognition of theS appropriate mRNA codon O
H H N H
•  All tRNAs are modified chemically, ex.
N inosine N
H
H N O N N H

P P
ribose ribose

sulfur replaces oxygen in U deamination of A

(4-thiouridine) (inosine)
 Reading frames
5’ ATGTTTGCTGACGGTTTAACGGAAGGCGGAAACATGGCGAAGAAAAAACCAGTAGAAAAA 3’
---------+---------+---------+---------+---------+---------+
3’ TACAAACGACTGCCAAATTGCCTTCCGCCTTTGTACCGCTTCTTTTTTGGTCATCTTTTT 5’

a M F A D G L T E G G N M A K K K P V E K
b C L L T V * R K A E T W R R K N Q * K K
c V C * R F N G R R K H G E E K T S R K K
---------+---------+---------+---------+---------+---------+
d H K S V T * R F A S V H R L F F W Y F F
e N A S P K V S P P F M A F F F G T S F
f T Q Q R N L P L R F C P S S F V L L F

6 reading frames (a–f) in dsDNA

(of course, only 3 in mRNA)

ORF = Open Reading Frame
From start codon to stop codon
Initiation Codon establishes the proper
“reading-frame”!
 tRNA

Wobble base-pairing
between codons and anticodons
anticodon

3′ 5′
wobble position
•  some aa have more than one tRNA
5′ 3′
codon and some tRNAs are constructed so
mRNA
that they require accurate base-pairing
bacteria
only at the first two positions of the
wobble codon possible
base anticodon bases codon and can tolerate a mismatch (or
U A, G, or I wobble) at the third position
C G or I

A U or I •  many of the alternative codons for an

G C or U amino acid differ only in their third
eukaryotes nucleotide
wobble codon possible
base anticodon bases

U A, G, or I

C G or I

A U

G C Figure 6-51
The Mechanism of Translation
Initiation in Prokaryotes
The Mechanism of Translation
Elongation in Prokaryotes (1)

E = exit site
P = peptidyl binding site
A = aminoacyl binding site
The Mechanism of Translation
Elongation in Prokaryotes (2)

Binding of a specific amino acid tRNA to

A site
The Mechanism of Translation
Elongation in Prokaryotes (3)

Peptide bond formation: chain

transfer from peptidyl tRNA to
aminoacyl tRNA
The Mechanism of Translation
Elongation in Prokaryotes (4)

Translocation of peptidyl tRNA

from A site to P site. Ribosome
moves one codon to the right, and
the now uncharged tRNA moves
from P site to E site.
The Mechanism of Translation
Elongation in Prokaryotes (5)

Ribosome is ready to start another cycle.

The cycles will continue until a
termination codon is reached.
The Mechanism of Translation
Termination in Prokaryotes
1. Initiation
•  Attachment of initiator
tRNA (Met-tRNA) to start
codon on mRNA and
assembly of ribosomal
subunits
•  Several initiation factors
assist the process (IF)
•  Strongly regulated
process
•  Uses energy form ATP or
GTP hydrolysis
2. Elongation
•  Repetitive cycles of codon directed addition of aa-tRNA
- AA-tRNA binding
- Proofreading
- Peptidyl transfer
- Translocation
2. Elongation (cont…)
•  AA-tRNA binding
- Elongation factor eEF-1
delivers amino acid-tRNA to
ribosome
- eEF-1 is bound to GTP

•  Proofreading
ü  AA-tRNA is checked against codon
ü  GTP hydrolysis and dissociation of eEF-1 from ribosome
ü  Correct amino acid-tRNA retained based on codonanticodon pairing
ü  Incorrect amino acid-tRNA escape from the ribosome
2. Elongation (cont…)
•  Peptidyl transfer
- Large rRNA catalyzes formation of peptide bond
- Precise orientation and stabilization of the transition
state
•  Translocation
- Binding EF-2-GTP
- Movement of peptidyl-tRNA to P Site
- Sliding of mRNA by 3 bases tRNA moved to the
exit
•  GTP hydrolysis and release of
EF-2
 3. Termination
•  Protein factor eTF binds to stop codon and catalyzes
hydrolysis of last amino acid-tRNA
•  Peptide is released from the ribosome
•  Ribosomal subunits dissociate

http://www.youtube.com/watch?v=-zb6r1MMTkc&feature=related
 has an anticodon that matches the amino acid. A second enzyme then chemically

Specific enzymes couple each AA to its appropriate

modifies each “incorrectly” attached amino acid so that it now corresponds to the
anticodon displayed by its covalently linked tRNA.
tRNA molecule: aminoacyl-tRNA synthetases
The synthetase-catalyzed reaction that attaches the amino acid to the 3 end of
the tRNA is one of many reactions coupled to the energy-releasing hydrolysis of
ATP (see pp. 64–65), and it produces a high-energy bond between the tRNA and
MBoC6 m6.55/6.53
the amino acid. The energy of this bond is used at a later stage in protein synthesis
to • 
link attaches aacovalently
the amino acid to the 3’ end
to the of the
growing tRNA à
polypeptide energy-releasing hydrolysis
chain.
The aminoacyl-tRNA synthetase enzymes and the tRNAs are equally important
of ATP process
in the decoding à high energy
(Figure 6–56).bond between
This was established tRNA
by an and aa338in Chapter 6: How Cells R
experiment

(A)
R OH aminoacyl-
O
tRNA
H2N C C
OH
H
ATP amino acid tRNA

R
P P O
H2N C C O O
P ribose adenine R C
H O
2 Pi H2N C C
adenylated amino acid H C R
O
H
NH2
aminoacyl-
tRNA Figure 6–55
P ribose adenine The structure of the
AMP which oneaminoacyl-tRNA
amino acid (cysteine) w
acid (alanine) after it already had
Figure 6–54
Figure 6–54 Amino Amino acid
acid activation activation
by synthetase by synthetase
enzymes. enzymes
An amino acid is activated for linkage
“hybrid” aminoacyl-tRNA molecu
protein synthesis by an aminoacyl-tRNA synthetase enzyme in two steps. As indicated, the energy
system,
of ATP hydrolysis is used to attach each amino acid to its tRNA molecule in a high-energy linkage. the wrong amino acid w
Coupling of amino acids to tRNAs
1. The amino acid is accepted by the
aminoacyl-tRNA synthetase enzyme
and is adenylated
2. The proper tRNA is accepted by
the enzyme and the amino acid
residue is transferred to the 2’ or
3’ OH of the 3’-terminal residue of
the RNA
Individual synthetase for each
amino acid and corresponding
tRNA(s)
the correct amino acid by a two-step mechanism. The correct amino acid has
the highest affinity for the active-site pocket of its synthetase and is therefore
The genetic code is translated by means
favored over the other 19; in particular, amino acids larger than the correct one
are excluded from the active site. However, accurate discrimination between two
of two adaptors that act one after another
similar amino acids, such as isoleucine and valine (which differ by only a methyl

amino acid
(tryptophan) H H H
O O O
H2N C C H2N C C high-energy H2N C C
OH O bond O
tRNA
Trp
CH2 (tRNA ) CH2 CH2
C C C
CH CH CH
N N N
H H H

ATP AMP + 2Pi

A C C 3′ A C C 5′
A C C linkage of amino acid tRNA binds to its base-pairing
to tRNA codon in RNA
tRNA synthetase U G G
(tryptophanyl 5′ 3′
tRNA synthetase)
mRNA

NET RESULT: AMINO ACID IS

SELECTED BY ITS CODON

Figure 6–56 The genetic code is translated by means of two adaptors that act one after another. The first adaptor is the aminoacyl-tRNA
synthetase, which couples a particular amino acid to its corresponding tRNA; the second adaptor is the tRNA molecule itself, whose
Figureanticodon
6-56
 Editing by tRNA synthetases ensures
accuracy FROM RNA TO PROTEIN

(A)
•  The correct amino acid editing site
incorrect
amino acid
tRNA
has the highest affinity 5′ 5′ 3′
will be
removed

for the active-site pocket 3′

synthesis
site
of its synthetase.
incorrect
•  After the amino acid has amino acid tRNA
synthetase
been covalently linked to
AMP: a second editing SYNTHESIZING EDITING
pocket: an amino acid is
removed from the AMP by (B) 5′
template
strand
hydrolysis: hydrolytic
3′
editing 5′ P
P
E E
newly
synthesized
•  1 m i s t a k e i n 4 0 , 0 0 0 DNA

couplings
POLYMERIZING EDITING

group), is very difficult to achieve in a single step. A second discrimination ste

occurs after the amino acid has been covalently linked to AMP Figure 6-57
(see Figure 6–54
 Amino acids are added to the C-terminal
end of a growing polypeptide chain
•  Formation peptide bond between carboxyl group at the end of a growing
polypeptide chain and a free amino group on an incoming amino acid
•  A protein is synthesized stepwise from its N-terminal end to its C-terminal
end
•  The growing carboxyl end of the polypeptide chain remains activated by its
340 Chapter 6: How Cells Read the Genome: From DNA to Protein
covalent attachment to a tRNA molecule (forming a peptidyl-tRNA)
R4 O
H O R2 H H O R4 O
H O R2 H H O H2N C C
H2N C C N C C N C C N C C
H2N C C N C C N C C O
H
O R1 H H O R3 H H O
R1 H H O R3

OH
aminoacyl-
peptidyl-tRNA attached to tRNA
C-terminus of the growing
polypeptide chain tRNA molecule freed from
4 its peptidyl linkage
4
3

3
new peptidyl-tRNA molecule
attached to C-terminus of
the growing polypeptide chain

Figure 6-59
Figure 6–59 The incorporation of an amino acid into a protein. A polypeptide chain grows by the stepwise addition of amino
ctors Drive Translation Forward and Improve Its STEP 3
2 3

Translating an mRNA molecule

1 4
H2N
polypeptide elongation shown in outline in Figure 6–64 has an
e that makes translation especially efficient and accurate. Two P A
growing 3 4
s enter and leave thepolypeptide
ribosome chain
during each cycle, each hydro-
STEP 1
P and undergoing conformational changes
newly bound
(1)
in the process. These (4) 5Small
′ subunit 3′
EF-Tu and1 EF-G 2 in bacteria, and EF1 and EF2 in eukaryotes. translocation
3 4 charged tRNA tRNA
H N
ditions in vitro, ribosomes can be forced to synthesize proteins
2

binding STEP 4
E P A
2
Figure 6–64 Translating3an mRNA 4 molecule. Each amino acid added to 3
1
the growing end of a polypeptide chain is selected by complementary base- H 2N 4
5′ 3′
pairing between the anticodon on its attached tRNA molecule and the next
codon on the mRNA chain. Because only one of the many types of tRNA 3
A
molecules in aE cell
site canPbase-pair
site A site
with each codon, the codon determines 4
ejected tRNA
the specific
STEPamino
2 acid to be added to the growing polypeptide chain. The
2
four-step cycle shown is repeated
3 (2) Peptide
over and over during the synthesis of a 5′ 3′
protein. In step1 1, an aminoacyl-tRNA molecule binds to a vacant A site on
H2N 4
bond
the ribosome. In step 2, a new peptide bond is formed. In step 3, the large STEP 1
subunit translocates relative to the small subunit, leaving the two tRNAs in
hybrid sites: P on theE largeP subunit
A
formation
and A on the small, for one; E on the 2 3
newly
bound
3 4 1 charged
large subunit and P on the small, for the other. In step 4, the small subunit H 2N 4 5
tRNA
translocates
5′
carrying its mRNA a distance of3′three nucleotides through the
ribosome. This “resets” the ribosome with a fully empty A site, ready for the
E
next aminoacyl-tRNA molecule to bind. As indicated, the mRNA is translated 4 5
peptidyl transferase
in the 5 -to-3 direction, and the N-terminal end of a protein is made first, with
STEP 3
each cycle adding one amino acid to the C-terminus of the polypeptide chain 5′ 3′
2
(Movie 6.7 and Movie
1
3
6.8).
4 (3) Large
H2N
subunit
3
P
4
A
translocation
5′ 3′
MBoC6 m6.66/6.64
Fig 6-64
 , the ribosome has begun the standard elongation cycle,
the start of translation. Instead, each bacterial mRNA contains a specific ribo- INITIATION FACTORS
E
6–64. P
some-binding site (called the Shine–Dalgarno sequence, named after its discov- DISSOCIATE A

Nucleotide sequences in mRNA signal

AAAAAAAA
LARGE
erers) that is located a few nucleotides upstream of the AUG at whicheIF4G translation RIBOSOMAL
is to begin. This nucleotide sequence, with the consensus 5 -AGGAGGU-3 , forms Met SUBUNIT
mRNA

where to start protein synthesis

BINDS
t. In 90% ofbase
mRNAs, pairstranslation
with the 16S rRNAatofthe
begins thefirst
small ribosomal subunit to position
eIF4E the
5′ cap initiat-
E A
ubunit. AtingthisAUG codon
point, the in the ribosome.
initiation factorsAdis- set of translation initiation factors
Met
orchestrates
additional 5′ 3′
mal subunit thistointeraction,
assemble with as well as the subsequent
the complex and assemblyGTP of the largeinitiation
ribosomalfactorssub- AUG
•  Start codon AUG and initiator tRNA (carries methionine)
unit to complete the ribosome.
ator tRNA remains at the P site, leaving the A site P
aa

Unlike a eukaryotic
ore ready to begin (see Figure 6–70). ribosome, a bacterial ribosome can readily mRNAassemble
•  Eukaryotes: the initiator tRNA - methionine complex (Met - tRNAi)
directly on a start codon
surrounding the start site in eukaryotic mRNAs that lies in the 5′
interior of an mRNA
AUG molecule, so3′long
ecognition as a ribosome-binding siteprocess.
precedes If it by several nucleotides.
AMINOACYL- As a result, bac-
is first loaded into the small ribosomal subunit along with Met
during
ntially fromterial
the above scanning
mRNAs are
the consensus often polycistronic—that
recognition sequence
additional
tRNA BINDS ATP
is, they encode several
aa (step 1)
INITIATOR tRNA
MOVESdifferent
ALONG pro-
RNA SEARCHING
proteins called eukaryotic initiation factors, or eIFs.
teins, each of which is
somal subunits will sometimes ignore the first
In contrast,
translated from the same Pi mRNA
+ ADP molecule (Figure
FOR FIRST AUG
6–71).
or third aAUG eukaryotic mRNA generally encodes only a single protein, or more E
p to the second codon instead. Cells
eIF2
, known asaccurately, a single to setproduce
of closely related
or proteins.
5′ 3′
“leaky scanning,” two Met
GTP AUG
Met
N-termini, from the GTPsame mRNA molecule. This
FIRST PEPTIDE
o produceStop Codons
theP same protein Mark withthe andEnd withoutof Translation P
BOND FORMS
initiator tRNA 5′ 3′ (step 2)
N-terminus,Theforend example, so that the protein
of the protein-coding message is is signaled AUG
by the presence of one of three
ments in thestopcell. small ribosomal
codonssubunit
(UAA, UAG, or UGA) (see Figure 6–48). These are not recognized by Met
with
g a start codon in bacteria is different. Bacte- Pi + GDP aa
a tRNA and do not specify an amino acid, but instead signal to the ribosome to
initiator tRNA
gnal the ribosome where
boundtotobegin
stop translation.
P site searching for
Proteins known as releaseeIF2 factors bind to any ribosome with a
AND OTHER
each bacterial mRNA contains
stop codon positioned a specific ribo- E
AAAAAAAA in the A site, forcing the peptidyl transferase in P the ribo-
INITIATION FACTORS
5′ 3′
ne–Dalgarno sequence, named
some to catalyze the eIF4G after its discov- DISSOCIATE
addition of a water molecule instead of an amino acid
A
to the AUG
LARGE
otides upstream of the AUG
peptidyl-tRNA at which
(Figure translation
6–72). This reaction frees the carboxyl endRIBOSOMAL of the growing
nce, with the consensuschain
polypeptide 5 -AGGAGGU-3 mRNA , forms Met SUBUNIT
eIF4E 5from
′ cap its attachment to a tRNA molecule, andBINDS since only this
e small ribosomal subunit to position the initiat-
attachment normally holds the growing polypeptide toE the ribosome, the com- etc.
A
A set of translation
Met
pleted initiation factors
additional orchestrates
GTP protein chain initiationis factors
immediately released 5′ into the cytoplasm. The ribosome 3′
bsequent assembly of the large ribosomal sub-
then releases its bound mRNA molecule and separates into AUG
the large and small
P
aa
subunits. These subunits
mRNAcan then assemble on this or another mRNA molecule
e, a5′ bacterial ribosome
to begin aAUG can readily 3′assemble
new round of protein synthesis.
s in the interior of an mRNA molecule, so long MBoC6 m6.72/6.70
edes it by several INITIATORAstRNA
ATP nucleotides.
MOVES ALONG
a result, bac- AMINOACYL-
tRNA BINDS
onic—thatPis,+ they
ADP
encode
RNAseveral different pro-
SEARCHING Met aa (step 1)
i
from the same mRNA molecule (Figure 6–71).
FOR FIRST AUG
enerally encodes only a single protein, or more E

related proteins. Met 5 3′

GTP ′
AUG
P
FIRST PEPTIDE
Fig 6-70
 Bacterial mRNAs are often polycistronic
•  Have no 5’ caps to signal the ribosome
•  A specific ribosome-binding site (Shine–Dalgarno sequence):
located a few nucleotides upstream of the AUG at which translation
is to begin (5’-AGGAGGU-3’)
•  Forms base pairs with the 16S rRNA of the small ribosomal subunit
to position the initiating AUG codon in the ribosome.
•  Bacterial mRNAs are often polycistronic: encode several different
proteins,
FROM RNA TO each of which is translated from the same mRNA
PROTEIN
molecule.

ribosome-binding sites
5′ 3′
P P P mRNA
AUG AUG AUG

protein α protein β protein γ

Figure 6–71 Structure of a typical bacterial mRNA molecule. Unlike eukaryotic ribosomes,
which typically require a capped 5 end on the mRNA, prokaryotic ribosomes initiate translation
Fig 6-71
 Structure of Prokaryotic mRNAs

mRNA has also regions that do not encode for a protein

Shine-Dalgarno sequence (SD) = Ribosome Binding Site (RBS)
The first AUG after SD-sequence is interpreted as the start site of translation
 Shine-Dalgarno Sequences
Help to align ribosomes on mRNA to properly start translation
Can base-pair with a sequence (ACCUCCUUA) contained in the ribosomal RNA
 ease the accuracy of this binding reaction, the ribosome and EF-Tu
and in
her EF-G,
thewhich drive translation
following ways. First, in the the 16s rRNA in the small subunit of

Elongation Factors (EFs) drive translation

he text, EF-Tu provides opportunities for EF-Tu P A
me assesses the “correctness”
on match. In this way, incorrectly paired
of the codon–anticodon match
A
P A
by folding
5′ 3′
nd probingofitstranslation
the accuracy molecular details (Figure
is improved. 6–66). When a correct match
he
to rRNA
the ribosome
gement
al change of the
forward and improve its accuracy
closesand tightly
inribosome
the ribosome
around the codon–anticodon
the subsequent
structure, moving
that
E site pair, causing
P site A site
triggers GTP hydrolysis by EF-Tu. Only
mRNAa con-
hree nucleotides
is• hydrolyzed through it (Movie
does EF-Tu release 6.9). its gripenter
on the aminoacyl-tRNA
2 elongation factors and leaveandthe
be used in protein synthesis. Incorrect codon–anticodon matches GTP do
ribosome during each
trigger this conformational change, and these errant tRNAs cycle, each hydrolyzing
mostly fall
P A
some
d GTPGTP before to GDP
they
hydrolysis, can
butbe thisused in protein synthesis. Proofreading, how-
synthe- P A

not end here.

oupling the GTP hydrolysis-driven
TP • 
ns between EF-Tu
is hydrolyzed and
and EF-Tu
different EF-G
states the (bacteria),
dissociates
of EF1 and
from the ribosome, incorrectlyEF2
there is abase-
paired tRNAs EF-G
portunity
ously. (eukaryotes)
The for theofribosome
cycles elongation to fac-
prevent an incorrect amino acid from
preferentially
GTP
ed alsoto the growing dissociate
on ensure thatchain.
all such There
changesis a shortPROOFREADING
time delay as the amino acid
the tRNA
ation to proceedmovesefficiently
into position (Figure on the ribosome. This time delay is
Pi
correct than incorrect codon–anticodon pairs. Moreover, incorrectly
GTP
RNAs
EF-Tudissociate
increases more rapidly As
its accuracy. than wethose correctly bound because their
withbind
ously the codon
GTP and is weaker.
aminoacyl-tR-Thus, most incorrectly bound tRNA GDP mole- GTP
EF-Tu A A A
ell as a significant
is form that the initialA
P
number
A
of correctly bound molecules) will leave the
codon–anti- P A E
P P

5′
without being
bosome. Because used protein3′synthesis. The two proofreading steps,
for free-energy
of the
eries, areE largely responsible mRNA for the 99.99% accuracy of the ribosome in
a correct codon–anticodon
site
P site A site
match GDP
RNA into
action. protein.this difference in
However, PROOFREADING
incorrectly base- Pi
lfthe wrong
account foramino
the high acid slips through
accuracy of the proofreading steps just
paired tRNAs
and is incorporated ontoGTP the growing polypeptide chain, there is still
preferentially GDP
opportunity dissociate
reaction, thefor the
ribosome
P
ribosome
A and EF-Tu to detect the error and provide a solu-
e one
16s rRNAthat isinnot, thestrictly
small subunitspeaking, of proofreading. An incorrectP A
codon‒
interaction in the match
codon–anticodon P site of bythe ribosome (which would occur after the
folding
incorrectly base-
oration)
gure 6–66). causesWhen an aincreased
correct paired
rate
match
tRNAs
of misreading in the A site. Succes-
E P A
s of amino acidpair,
don–anticodon misincorporation
causing a con- eventually lead to premature ter-
preferentially
ofersthe protein
PROOFREADING by release dissociate
factors, which are described below. Normally,
GTP hydrolysis by EF-Tu. Only
ssegripfactors
on the actaminoacyl-tRNA
when translationand of a protein is complete; here, they act
ough this mechanism does
rect codon–anticodon
Pi
matches not correct
do the original error, it releases the Fig 6-65
 walls of this tunnel, mademRNA primarily of 23S rRNA, A SITE
Metare a patchwork
Trp of tiny hydro-
phobicAUGsurfaces embedded in a more extensive hydrophilic surface. This struc- E P A
H 2N
ture is not complementary to any peptide, and thus provides a “Teflon” coating
Stop codons mark the end of translation
ACC
through which a polypeptide chain can easily slide. The AUGAACUGGUAGCGAUCG
E dimensions
P A of the tunnel
protein β suggest that protein
nascent proteins are largely unstructured as they pass through the
γ 5′ 3′
ACC
ribosome,
al mRNA molecule. although
Unlike some
eukaryotic -helical regions of the
ribosomes, A Uprotein
G A A C U Gcan
G U Aform
G C G Abefore
UCG leaving
•  One
the ribosomeof three
tunnel. As stop
it leaves
he mRNA, prokaryotic ribosomes initiate translation codons
the ribosome, 5′ a(UAA,
newly UAG,
synthesized or
protein
3′ UGA):
must not recognized
HO 2
by a
equences),fold into its proper three-dimensional conformation to be useful to the cell. Later
which can be located anywhere along an
intRNA toand domorenot specify an First,
amino acid, signalsev- to the ribosome to stop
TERMINATION
omes permits bacteria synthesize than one COOH
this chapter we discuss how this folding occurs. however, we describe
e. Trp
translation
eral additional aspects of the translation process itself.
Asn

•  Proteins
m6.73/6.71
Proteins known
Are Made as releaseMetfactors
on Polyribosomes Asn
Trp bind
BINDING OF
RELEASE
to any ribosome
Met with a stop
olypeptide moves through a large, water-filled
nm) in thecodon
The synthesispositioned
large subunit of the ribosome.in The the A between
site, forcing theseveral
peptidyl transferase in the
of most protein molecules takes 20 seconds and
FACTOR
HN TO THE
minutes. During this very short period, however, it is usual for multiple
2
initiations NH
349hydro-
toribosome to ofcatalyze the addition of a water molecule instead of an
A SITE 2
y of 23S rRNA, are a patchwork tiny
take place on each mRNA molecule being translated. As
E P A soon as the preceding
ore extensive hydrophilic surface. This struc-
ribosome has translated enough of the nucleotide sequence to move out of the P A A
eptide, andamino acid
thus provides to the
a “Teflon” peptidyl-tRNA.
coating ACC
way, the 5 end of the mRNA is threaded into a new ribosome. The mRNA mole-
E
can easily slide.AsnThe dimensions of the tunnel AUGAACUGGUAGCGAUCG
cules beingTrp translated are therefore usually found 5 ′ in the form of polyribosomes
3′ (or ACC
argely unstructured
Met as they pass through the AUGAACUGGUAGCGAUCG
polysomes): large cytoplasmic assemblies made up of several ribosomes spaced 5′ 3′
regions of the protein can form before leaving
H2N as close as 80 nucleotides apart along a single mRNA molecule (Figure 6–73).
e ribosome, a newly synthesized protein must H2more
O
These multiple initiations allow the cell to make many protein molecules in
al conformationE to be P useful
A to the cell. Later
a given time than would be possible if each protein had to beTERMINATION
COOH completed before DISSOCIATION
olding occurs. First, however, we describe sev-
the next could ACC start. Trp
ation process Uitself.
ABoth
G A A bacteria
C U G G U A Gand
C G A Ueukaryotes
CG use polysomes, Asn and both employ additional
strategies to speed up the3′ overall rate of protein synthesis. Because bacterial
5′
osomesmRNA does not need to be processed and is accessible Met to ribosomes while it is A C
P AC
being
cules takes made, ribosomes
between 20 secondscan andattach
severalto the free end of a bacterial mRNA molecule E
od, however, it is usual for multiple initiations transcription
and start translating it even before the NH2 of that RNA is complete, fol-
ule beinglowing closely
translated.
Asn Asbehind
soon asthe theRNA polymerase as it moves along DNA. In eukaryotes,
preceding AUGAACUGGUAGCGAUCG
as we have seen, the BINDING
5 and 3 OF
ends of the mRNA interact (see Figure 6–73A); there- 5′ 3′
the nucleotide
Met sequence Trp to move
RELEASEout of the P A A
E
aded into fore, as soon
a new as a ribosome
ribosome. The mRNA
FACTOR dissociates,
mole- its two subunits are in an optimal position
H2N to reinitiate translation TO onTHEthe same mRNA molecule. ACC
usually found in the form of polyribosomes
A SITE (or AUGAACUGGUAGCGAUCG
mblies made upEof several P A ribosomes spaced 5′ 3′
There
ong a single mRNAAre molecule
Minor Variations
(Figure 6–73). in the Standard Genetic Code
cell to make many ACC more protein molecules in
AsAdiscussed
U G A A C U G Gin
U AChapter
G C G A U C G1, the genetic code (shown in Figure 6–48) applies to
Figure 6–72 The final phase of protein
e if each5protein
′ three had to be completed before
all major branches of 3′ life, providing important evidence for the common
DISSOCIATION
synthesis. The binding of a release Fig 6-72
 Proteins are made on polyribosomes
•  The mRNA molecules being translated are usually found in
the form of polyribosomes (or polysomes): large
cytoplasmic assemblies made up of several ribosomes
spaced as close as 80 nucleotides apart along a single
mRNA molecule.
•  Bacterial mRNA does not need to be processed and is
accessible to ribosomes while it is being made, ribosomes
can attach to the free end of a bacterial mRNA molecule and
start translating it even before the transcription of that RNA
is complete, following closely behind the RNA polymerase
as it moves along DNA.
•  In eukaryotes, the 5’ and 3’ ends of the mRNA interact, as
soon as a ribosome dissociates, its two subunits are in an
optimal position to reinitiate translation on the same mRNA
molecule.
 350 Chapter 6: How Cells Read the Genome: From DNA to Protein

Proteins are made on polyribosomes messenger RNA

3′ (mRNA)
AA
AA
A
A AA eIF4G
A
5′ 5′ cap
G
UA

AU
eIF4E

G
stop start
codon codon
poly-A-binding
protein

growing
polypeptide
chain
100 nm 100 nm
(A) (B)

cytosol of the cell it is translated as isoleucine (see Table 14–3, p. 805). This type o
deviation in the genetic code is “hardwired” into the organisms or the organelle
in which it occurs.
A different type of variation, sometimes called translation recoding, occur
in many cells. In this case, other nucleotide sequence information present in an
mRNA can change the meaning of the genetic code at a particular site in the mRNA
molecule. The standard code allows cells to manufacture proteins using only 2
amino acids. However, bacteria, archaea, and eukaryotes have available to them
twenty-first amino acid that can be incorporated directly into a growing polypep
tide chain through translationMBoC6recoding. Fig 6-72
Selenocysteine, which is essential
m6.76/6.73
for th
POLISOM

60
POLISOM

61
 Inhibitors of prokaryotic protein
352
synthesis are useful as antibiotics
Chapter 6: How Cells Read the Genome: From DNA to Protein

TABLE 6–4 Inhibitors of Protein or RNA Synthesis

Inhibitor Specific effect
Acting only on bacteria
Tetracycline Blocks binding of aminoacyl-tRNA to the A site of ribosome
Streptomycin Prevents the transition from translation initiation to chain elongation and also causes miscoding
Chloramphenicol Blocks the peptidyl transferase reaction on ribosomes (step 2 in Figure 6–64)
Erythromycin Binds in the exit channel of the ribosome and thereby inhibits elongation of the peptide chain
Rifamycin Blocks initiation of RNA chains by binding to RNA polymerase (prevents RNA synthesis)
Acting on bacteria and eukaryotes
Puromycin Causes the premature release of nascent polypeptide chains by its addition to the growing chain end
Actinomycin D Binds to DNA and blocks the movement of RNA polymerase (prevents RNA synthesis)
Acting on eukaryotes but not bacteria
Cycloheximide Blocks the translocation reaction on ribosomes (step 3 in Figure 6–64)
Anisomycin Blocks the peptidyl transferase reaction on ribosomes (step 2 in Figure 6–64)
-Amanitin Blocks mRNA synthesis by binding preferentially to RNA polymerase II
The ribosomes of eukaryotic mitochondria (and chloroplasts) often resemble those of bacteria in their sensitivity to inhibitors. Therefore, some of
these antibiotics can have a deleterious effect on human mitochondria.
Protein synthesis
1.  Initiation
2.  Elongation
3.  Termination
 Protein sintesis pada eukariot
1.  Asam amino awal adalah METIONIN,
2.  Untuk mengikat tRNA fMet (awal) diperlukan 9 IF untuk
membentuk PRE INISIASI KOMPLEKS.
3.  Ikatan dengan IF dg tRNAinit harus ada sebelum Ikaan
IF dg mRNA ( pada Prok boleh sesudahnya).
4.  Lebih banyak IF untuk mengikat Ribosom 60s
5.  Empat EF
–  EF.1.α = EF-Tu (pd Prokariot).
–  EF.1.ß = EF-Ts
–  EF.1.Υ
–  EF.2 = EF.G.
6.  Transkripsi dan Translasi tidak pernah terjadi
bersamaan, karena Transkripsi terjadi dlm Nukleus,
Translasi terjadi diluar Nukleus.
7.  Waktu hidup mRNA Prokariot pendek (beberapa menit),
Eukariot sampai beberapa hari.
 What happens after translation?
•  A newly synthesized polypeptide chain must undergo post-
translational processing to generate the final protein
•  Posttranslational modifications include:
- Targeting to the appropriate cell compartment
- Folding
- Addition of sugar chains
- Formation of disulfide bonds
•  Posttranslational modifications start as soon as the nascent
polypeptide emerges from the tunnel in large ribosomal
subunit
- That means these changes happen during translation
when the peptide has NOT been finished yet and is still
attached to the ribosome (cotranslationally)
 How to detect gene expression?
1.  mRNA level
•  Northern blot (RNA-RNA)
•  Southern blot (DNA-DNA)
•  PCR (week 2)

2.  Protein level: antigen-antibody reaction

•  Immunostaining
•  Western blot
•  ELISA
 Northern/Southern/Western blot
Step:
1.  DNA/RNA/protein isolation
(RNA reverse transcripted à cDNA)
2.  Gel electrophoresis
3.  Transfer into a membrane (blotting)
4.  Detection:
•  Northern/Southern: probe (labeled DNA)
•  Western: antibody

Principle:
•  Northern/Southern: hybridization between DNA/RNA
sample and probe
•  Western: antigen-antibody binding
Example: α-tubulin expression

PCR

(Zhang et al, 2014, Chinese Journal of Cancer Research)

Example: α-tubulin expression

Western blot

(Abcam)
Example: α-tubulin expression

Immunostaining

(Abcam)
 ELISA
•  various antigen-antibody combinations are used,
always including an enzyme-labeled antigen or
antibody, and enzyme activity is measured
colorimetrically.
•  The enzyme activity is measured using a substrate that
changes color when modified by the enzyme. Light
absorption of the product formed after substrate
addition is measured and converted to numeric values.
•  Depending on the antigen-antibody combination, the
assay is called a direct ELISA, indirect ELISA, sandwich
ELISA, competitive ELISA etc.
http://ruo.mbl.co.jp/bio/e/support/method/
elisa.html
 References

Alberts, B. et al, 2015, Molecular Biology of the Cell, 6th

Edition, Garland Publishing, USA.

Sismindari et al, 2010, Translasi, Bahan Ajar Biologi

Molekuler (ppt), Departemen Kimia Farmasi,
Fakultas Farmasi UGM, Yogyakarta.

Selected articles.
 Animasi
•  Translasi di prokaryot
•  Translasi di eukaryot
Thank you