11

Protein Synthesis:
Translation of the
Genetic Message

© 2003 Thomson Learning, Inc.

All rights reserved
11-1
11 Protein Biosynthesis
A flow chart for protein biosynthesis

© 2003 Thomson Learning, Inc.

All rights reserved
11-2
11 The Genetic Code
• Features of the genetic code
• triplet: a sequence of three bases (a codon) is needed
to specify one amino acid
• nonoverlapping: no bases are shared between
consecutive codons
• commaless: no intervening bases between codons
• degenerate: more than one triplet can code for the
same amino acid; Leu, Ser, and Arg, for example, are
each coded for by six triplets
• universal: the same in viruses, prokaryotes, and
eukaryotes; the only exceptions are some codons in
mitochondria

© 2003 Thomson Learning, Inc.

All rights reserved
11-3
11 The Genetic Code
Nonoverlapping and overlapping codes

© 2003 Thomson Learning, Inc.

All rights reserved
11-4
11 The Genetic Code
• All 64 codons have been assigned
• 61 code for amino acids
• 3 (UAA, UAG, and UGA) serve as termination signals
• only Trp and Met have one codon each
• the third base is irrelevant for Leu, Val, Ser, Pro, Thr,
Ala, Gly, and Arg
• the second base is important for the type of amino
acid; for example, if the second base is U, the amino
acids coded for are hydrophobic
• for the 15 amino acids coded for by 2, 3, or 4 triplets, it
is only the third letter of the codon that varies. Gly, for
example, is coded for by GGA, GGG, GGC, and GGU
© 2003 Thomson Learning, Inc.
All rights reserved
11-5
11 The Genetic Code 5' U C A G 3'
UUU Phe UCU S er UAU Tyr UGU Cys U
UUC Phe UCC S er UAC Tyr UGC Cys C
U UUA Leu UCA S er UAA S top UGA S top A
UUG Leu UCG S er UAG S top UGG Trp G
CUU Leu CCU Pro CAU His CGU Arg U
CUC Leu CCC Pro CAC His CGC Arg C
C CUA Leu CCA Pro CAA Gln CGA Arg A
CUG Leu CCG Pro CAG Gln CGG Arg G
AUU Ile ACU Thr AAU Asn AGU S er U
AUC Ile ACC Thr AAC Asn AGC S er C
A
AUA Ile ACA Thr AAA Lys AGA Arg A
AUG Met* ACG Thr AAG Lys AGG Arg G
GUU Val GCU Ala GAU Asp GGU Gly U
GUC Val GCC Ala GAC Asp GGC Gly C
G GUA Val GCA Ala GGA Gly A
GAA Glu
GUG Val GCG Ala GAG Glu GGG Gly G
*AUG signals translation initiation as well as coding for Met
© 2003 Thomson Learning, Inc. 3rd base irrelevant Pyrimidines Unique definition
All rights reserved
Purines 3 out of 4 Unique definition 11-6
11 The Genetic Code
• Assignments of triplets is based on several
types of experiments
• synthetic mRNA: if mRNA is polyU, polyPhe is formed;
if mRNA is poly ---ACACAC---, poly(Thr-His) is formed
• binding assay: aminoacyl-tRNAs bind to ribosomes in
the presence of trinucleotides
• synthesize trinucleotides by chemical means
• carry out a binding assay for each type of
trinucleotide
• aminoacyl-tRNAs are tested for their ability to bind
in the presence of a given trinucleotide
• see figure, next screen
© 2003 Thomson Learning, Inc.
All rights reserved
11-7
11 Wobble Base Pairing
• Many tRNAs can recognize more than one codon
because of variations in allowed patterns of
hydrogen bonding
• the variation is called “wobble”
• wobble is in the first base of the anticodon
• base-pairing combinations in the wobble scheme are:
Base at 5' end Base at 3' end
of anticodon of codon
I* A, C, or U
G C or U
U A or G
A U
C G

© 2003 Thomson Learning, Inc.

I* = hypoxanthine
All rights reserved
11-8
11 Wobble Base Pairing
N H
O R N N
H
N N N N O
R H
Ade nosine H
O O N N
Uridine
H N
N Inosine N N
N R
N H
Inosine
R N
H
N N O
R
H N
O N
Cytidine
N N
Inosine R
© 2003 Thomson Learning, Inc.
All rights reserved
11-9
11 Wobble Base Pairing
• The wobble hypothesis provides insight into
some aspects of the degeneracy of the code
• in many cases, the degenerate codons for a given
amino acid differ only in the third base; therefore
fewer different tRNAs are needed because a given
tRNA can base-pair with several codons

• the existence of wobble minimizes the damage that

can be caused by a misreading of the code; for
example, if the Leu codon CUU were misread CUC or
CUA or CUG during transcription of mRNA, the codon
would still be translated as Leu during protein
synthesis
© 2003 Thomson Learning, Inc.
All rights reserved
11-10
11 Transfer RNA (tRNA)
• Acts as the adapter molecule between the
genetic code on mRNA and the protein
“language”
• 75-85 bases long
• A specific amino acid is covalently linked at
the 3’ end
• Elsewhere on the molecule is an anticodon
complimentary to the specific amino acid
codon on mRNA that codes for the amino acid
carried by the tRNA
• Contain a number of modified bases
© 2003 Thomson Learning, Inc.
All rights reserved
11-11
11 Transfer RNA (tRNA)
A Amino Acid attachment site
Acceptor Arm - A C
C
specific amino acid is 1
73
72
TyC arm - y stands
attached to the 3’ end 2
3
71
70
for pseudouridine
4 69
5 68
6 67
U* 7 66 Py 59A*
16 Pu 65 64 63 62 C
17 A 9 Pu
17:1
13 12 Py 10 49 50 51 52 G T C
G* Py
y
G 22 23 Pu 25 47:16

2020:120:2A 26 47:15
27
1 43 44
28 42 45
D Arm - Contains 29
30
41 46
40 47 Extra Arm - May
dihydrouridine
47:1
31 39
Py* 38 vary in size
U Pu*
34 35 36

Anticodon
© 2003 Thomson Learning, Inc.
All rights reserved
11-12
11 Aminoacyl-tRNA Synthetase
• Aminoacyl-tRNA Synthetase enzymes
attach the correct amino acids to the
correct tRNA
• This is an energy consuming process
• Aminoacyl-tRNA Synthetases
recognize tRNAs on the basis of their
looped structure, not by direct
recognition of the anticodon
© 2003 Thomson Learning, Inc.
All rights reserved
11-13
11 Amino-
Making
Gly acyl-tRNA
Synthetase
Aminoacyl-
P tRNA
P Amino-
ATP
P Gly acyl-tRNA
Synthetase
P
P
P Pyrophosphate
Amino-
Gly acyl-tRNA
Synthetase
P

CCA

© 2003 Thomson Learning, Inc.

All rights reserved
11-14
11 Amino-
Making
Gly acyl-tRNA
Synthetase
Aminoacyl-
P tRNA
P Amino-
ATP
P Gly acyl-tRNA
Synthetase
P
P
P Pyrophosphate
Amino-
Gly acyl-tRNA
Synthetase

Gly
Amino- P
acyl-tRNA AMP
Synthetase

CCA

Aminoacyl- Note that the amino acid is not paired with the
tRNA tRNA on the basis of the anticodon. The correct
© 2003 Thomson Learning, Inc.
tRNA for a given amino acid is recognized on
All rights reserved CCA the basis of other parts of
11-15
the molecule. ©1998 Timothy G. Standish
 O
11 H
Aminoacylation of tRNA
H N C

C O H
H O H O H

R H
C C
H
H
H
O H H C C
H O N H
N
C C C

3’ H C
O O
C N
N
5’
© 2003 Thomson Learning, Inc.
H O
P

O
C

All rights reserved

N 11-16
H H
 Aminoacylation Class I Aminoacyl
11 of tRNA
H
O tRNA Synthetases
attach amino acids to
H N C
the 2’ carbon while
Amino Class II attach to
C O H O H
acid the 3’carbon

tRNA
R H C C
H
H
H
O H H C C
H O N H
N
C C C

3’ H C
O O
C N
N
5’
© 2003 Thomson Learning, Inc.
H O
P

O
C

All rights reserved

N 11-17
H H
11 Classification of
Aminoacyl-tRNA Synthetases
Aminoacyl-tRNA Class I - 2’ OH Class II - 3’ OH
Synthetases (ARS) • Glu (a) • Gly (a b2
may be mono or • Gln (a) • Ala (a4
multimeric. • Arg (a) • Pro (a
Two types of • Val (a) • Ser (a
polypeptide chains • Ile (a) • Thr (a
• Leu (a)
are recognized: • Asp (a ??
• Met (a
a and b. • Asn (a
• Tyr (a • His (a
• (a • Lys (a
© 2003 Thomson Learning, Inc.
All rights reserved
11-18
11 Requirements for Translation
• Ribosomes - rRNA and Protiens
• mRNA - Nucleotides
• tRNA
• The RNA world theory might explain these three
components
• Aminoacyl-tRNA Synthetase,
• A protein, thus a product of translation and cannot
be explained away by the RNA world theory
• L Amino Acids
• ATP - For energy
• This appears to be an irreducibly complex system

© 2003 Thomson Learning, Inc.

All rights reserved
11-19
11 Amino Acid Activation
• Requires
• amino acids
• tRNAs
• aminoacyl-tRNA synthetases
• ATP, Mg2+
• Formation on an aminoacyl-tRNA

amino acid + ATP aminoacyl-AMP + PP i

aminoacyl-AMP + tRNA aminoacyl-tRNA + AMP

amino acid + ATP + tRNA

aminoacyl-tRNA + AMP + PP i

© 2003 Thomson Learning, Inc.

All rights reserved
11-20
11 Amino Acid Activation
S tep 1:
O O O O PP i O O
A -r ib- O- P- OPOPO - + - O- C- CH- R A -r ib- O- P- O- C-CH- R
O- O- O- N H3 + O- N H3 +
ATP Amino acid An aminoacyl-AMP

t RNA A
S tep 2: O
t RNA AMP
O O A H H
O
A -r ib- O- P- O- C-CH- R + H H H H
O- N H3 + H O HO
H
OH HO O C-CH- R
An aminoacyl-AMP
Transfer RNA
N H3 +
An aminoacyl-tRNA

© 2003 Thomson Learning, Inc.

All rights reserved
11-21
11 Amino Acid Activation
• This two-stage reaction allows selectivity at two
levels
• the amino acid: the aminoacyl-AMP remains bound to
the enzyme and binding of the correct amino acid is
verified by an editing site in the tRNA synthetase
• tRNA: there are specific binding sites on tRNAs that
are recognized by aminoacyl-tRNA synthetases.

© 2003 Thomson Learning, Inc.

All rights reserved
11-22
11 Amino Acid Activation
Ribbon diagram of tRNA tertiary structure

© 2003 Thomson Learning, Inc.

All rights reserved
11-23
11 Chain Initiation
• Requires
• fmet-tRNAfmet
• initiation codon (AUG) of mRNA
• 30S ribosomal subunit
• 50S ribosomal subunit
• initiation factors IF-1, IF-2, and IF-3
• GTP, Mg2+

© 2003 Thomson Learning, Inc.

All rights reserved
11-24
11

© 2003 Thomson Learning, Inc.

All rights reserved
11-25
11 Chain Initiation
• In prokaryotes, the initial N-terminal amino acid is
N-formylmethionine (fmet)
Met-tRNA
synthetase
Met + tRNA f met Met-tRNA f met
(ATP)
f met
Formyl- FH4
Met-tRNA
formyltransferase FH4

O
CH3 -S-CH2 CH2 CHC- t RN A
NH
H C O
f met
N-Formylmethionine-tRNA

© 2003 Thomson Learning, Inc.

All rights reserved
11-26
11 Chain Initiation
• both tRNAmet and tRNAfmet contain the triplet 3’-UAC-5’
• this triplet base pairs with 5’-AUG-3’ in mRNA
• the 3’-UAC-5’ triplet on tRNAfmet recognizes the AUG
triplet (the start signal) when it occurs at the beginning
of the mRNA sequence that directs polypeptide
synthesis
• the 3’-UAC-5’ triplet on tRNAmet recognizes the AUG
triplet when it is found in an internal position in the
mRNA sequence
• the start signal is preceded by a Shine-Dalgarno
purine-rich leader segment, 5’-GGAGGU-3’, which
usually lies about 10 nucleotides upstream of the AUG
start signal and acts as a ribosomal binding site
© 2003 Thomson Learning, Inc.
All rights reserved
11-27
11 Chain Initiation
• The start of polypeptide synthesis requires an
initiation complex composed of
• mRNA
• 30S ribosomal subunit
• fmet-tRNAfmet
• GTP
• IF-3; facilitates binding of mRNA to the 30S subunit
• IF-2; binds GTP and aids in selection of fmet-tRNAfmet
• IF-1; appears to facilitate binding of IF-3 and IF-2
• 50S ribosomal subunit
• The binding of these units produces the 70S
initiation complex
© 2003 Thomson Learning, Inc.
All rights reserved
11-28
11 Chain Initiation
Formation of an initiation complex

© 2003 Thomson Learning, Inc.

All rights reserved
11-29
11 Chain Elongation
• Requires
• 70S ribosome
• codons of mRNA
• aminoacyl-tRNAs
• elongation factors EF-Tu, EF-Ts, and EF-G
• GTP, and Mg2+

© 2003 Thomson Learning, Inc.

All rights reserved
11-30
11 Chain Elongation
Sine-Delgano sequences recognition by E. coli

© 2003 Thomson Learning, Inc.

All rights reserved
11-31
11 Chain Elongation
• Step 1
• an aminoacyl-tRNA is bound to the A site
• the P site is already occupied
• Step 2
• EF-Tu is released in a reaction requiring EF-Ts
• Step 3
• the peptide bond is formed, the P site is uncharged
• Step 4
• the uncharged tRNA is released
• the peptidyl-tRNA is translocated to the P site
• EF-G and GTP are required
• the
© 2003 Thomson
All rights reserved
next
Learning, Inc. aminoacyl-tRNA occupies the empty A site
11-32
11

© 2003 Thomson Learning, Inc.

All rights reserved
11-33
11 Chain Elongation
Adenine Adenine
t RNA f m e t t RNA
O O
H H H H
H H H H
O O HO O HO
H- C-N H- CH C= O H2 N -CH C= O
CH3 SCH2 CH2 R
N-Formylmethionine-tRNA f met An aminoacyl-tRNA

peptidyl transferase
fmet Adenine Adenine
t RNA t RNA
O O
H H + H H
H H H H
OH HO O O O HO
H- C-N H- CH -C- NH- CH C= O
© 2003 Thomson Learning, Inc. CH3 SCH2 CH2 R
All rights reserved
11-34
11 Chain Elongation
• puromycin
H3 C CH3
N N H2

N N N N

N O N
N N
HO- CH 2 t RNA -OPO- CH 2
O O
H H O- H H
H H H H
NH HO O HO
O C-CH- CH2 OCH3 O C-CH- R
N H3 + N H3 +
Puromycin An aminoacyl-tRNA

© 2003 Thomson Learning, Inc.

All rights reserved
11-35
11

© 2003 Thomson Learning, Inc.

All rights reserved
11-36
11

© 2003 Thomson Learning, Inc.

All rights reserved
11-37
11 Chain Termination
• Chain termination requires
• termination codons (UAA, UAG, or UGA) of mRNA
• RF-1 which binds to UAA and UAG or RF-2 which
binds to UAA and UGA
• RF-3 which does not bind to any termination codon,
but facilitates the binding of RF-1 and RF-2
• GTP which is bound to RF-3
• The entire complex dissociates setting free the
completed polypeptide, the release factors,
tRNA, mRNA, and the 30S and 50S ribosomal
subunits

© 2003 Thomson Learning, Inc.

All rights reserved
11-38
11 Protein Synthesis
• Summary of required components
S tep Components
Amino acid Amino acids, tRNAs, ATP, Mg 2+,
activation and aminoacyl-tRNA synthetases

Chain initiation fmet-tRNA f met, initiation codon (AUG),

Chain elongation
Chain termination
© 2003 Thomson Learning, Inc.
All rights reserved
2+
30S and 50S ribosomal subunits, GTP, Mg ,
and initiation factors (IF-1, IF-2, and IF-3)
2+
70S ribosome, codons for mRNA, GTP, Mg ,
elongation factors (EF-Tu, EF-Ts and EF-G),
and aminoacyl-tRNA
70S ribosome, termination codons (UAA,
UAG, and UGA), release factors (RF-1, RF-2,
and RF-3), GTP, and Mg 2+
11-39
11 Protein Synthesis
• in prokaryotes, translation begins very soon after
mRNA transcription
• it is possible to have several molecules of RNA
polymerase bound to a single DNA gene, each in a
different stage of transcription
• it is also possible to have several ribosomes bound to
a single mRNA, each in a different stage of translation
• polysome: mRNA bound to several ribosomes
• coupled translation: the process in which a
prokaryotic gene is being simultaneously transcribed
and translated

© 2003 Thomson Learning, Inc.

All rights reserved
11-40
11 Protein Synthesis
Peptide chain termination

© 2003 Thomson Learning, Inc.

All rights reserved
11-41
11

© 2003 Thomson Learning, Inc.

All rights reserved
11-42
11 Protein Synthesis
Simultaneous protein synthesis on polysomes

© 2003 Thomson Learning, Inc.

All rights reserved
11-43
11

© 2003 Thomson Learning, Inc.

All rights reserved
11-44
11 Translation - Eukaryotes
Structure of eukaryotic mRNAs

© 2003 Thomson Learning, Inc.

All rights reserved
11-45
11 Translation - Eukaryotes
• Chain elongation
• uses the same mechanism of peptidyl transferase and
ribosome translocation as prokaryotes
• there is no E site on eukaryotic ribosomes, only A and
P sites
• there are two elongation factors, eEF-1 and eEF-2
• Chain termination
• stop codons are the same: UAG, UAA, and UGA
• only one release factor that binds to all three stop
codons

© 2003 Thomson Learning, Inc.

All rights reserved
11-46
11 Polypeptide Modification
• Newly synthesized polypeptides are frequently
modified before they reach their final form
• N-formylmethionine in prokaryotes is cleaved
• specific bonds in precursors are cleaved, as for
example, preproinsulin to proinsulin to insulin
• leader sequences are removed by specific proteases
of the endoplasmic reticulum; the Golgi apparatus
then directs the finished protein to its final destination
• factors such as heme groups may be attached
• disulfide bonds may be formed
• amino acids may be modified, as for example,
conversion of proline to hydroxyproline
• other
© 2003 Thomson covalent modifications; e.g., addition of
Learning, Inc.
All rights reserved
carbohydrates 11-47
11

© 2003 Thomson Learning, Inc.

All rights reserved
11-48
11 Viruses, Cancer, and AIDS
• as the result of the accumulation of large-T protein,
the infected cell behaves like a cancer cell
• the large-T gene is an oncogene, a gene that causes
cancer
• Oncogenes
• are transported from one cell to another by viruses,
but they normally originate in cells that have been
infected by the virus at some earlier time
• analogues of these genes, called protooncogenes,
exist in normal cells in many different species, cells
that have not been infected by a virus
• when some triggering event takes place, the
analogues are transformed
© 2003 Thomson Learning, Inc.
All rights reserved
11-49
11 Viruses, Cancer, and AIDS
• as a result of the transformation, the cell becomes a
cancer cell
• viral oncogenes have been incorporated into the
genomes of many viruses and are carried along with
the genes needed for replication of the virus
• many viruses that transmit oncogenes are
retroviruses, RNA viruses that make use of reverse
transcriptase to copy their RNA genomes into DNA
• the RNA genomes of all retroviruses have genes for
coat proteins (CP), for reverse transcriptase (RT), and
envelope protein (EP)

© 2003 Thomson Learning, Inc.

All rights reserved
11-50
11 Viruses, Cancer, and AIDS
The architecture of HIV

© 2003 Thomson Learning, Inc.

All rights reserved
11-51
11 Viruses, Cancer, and AIDS
Infection of cells by simian virus 40

© 2003 Thomson Learning, Inc.

All rights reserved
11-52
11 Viruses, Cancer, and AIDS
RNA genomes of retroviruses

© 2003 Thomson Learning, Inc.

All rights reserved
11-53
11 Viruses, Cancer, and AIDS
Beginning of HIV infection

© 2003 Thomson Learning, Inc.

All rights reserved
11-54