The document discusses protein synthesis and the genetic code. It describes the key components involved in translation including mRNA, tRNA, ribosomes, aminoacyl-tRNA synthetases, and initiation factors. The genetic code is triplet-based, with 61 codons coding for 20 amino acids. Wobble base pairing and degeneracy in the code allow one tRNA to recognize multiple codons. Aminoacyl-tRNA synthetases attach the correct amino acid to the correct tRNA. Initiation involves the formation of fMet-tRNA which binds to the start codon on mRNA.

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Protein Synthesis:
Translation of the
Genetic Message

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Protein Biosynthesis
A flow chart for protein biosynthesis

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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

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The Genetic Code
Nonoverlapping and overlapping codes

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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

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The Genetic Code
A
UCU
UCC
UCA
UCG
U C G
U
C
A
G
CUU
CUC
CUA
CUG
CCU
CCC
CCA
CCG
CGU
CGC
CGA
CGG
U
C
A
G
A
G
ACU
ACC
ACA
ACG
U
C
A
G
U
C
A
G
GUU
GUC
GUA
GUG
GCU
GCC
GCA
GCG
GGU
GGC
GGA
GGG
Leu
Leu
Leu
Leu
Val
Val
Val
Val
Ser
Ser
Ser
Ser
Pro
Pro
Pro
Pro
Thr
Thr
Thr
Thr
Ala
Ala
Ala
Ala
Stop
Arg
Arg
Arg
Arg
Gly
Gly
Gly
Gly
5' 3'
U
C
UUU
UUC
Phe
Phe
UUA
UUG
Leu
Leu
Ile
Ile
Ile
AUU
AUC
AUA
Met*
AUG
UAU
UAC
Tyr
Tyr
UAA
UAG
Stop
Stop
CAU
CAC
His
His
CAA
CAG
Gln
Gln
AAU
AAC
Asn
Asn
AAA
AAG
Lys
Lys
GAU
GAC
Asp
Asp
GAA
GAG
Glu
Glu
UGA
Trp
UGG
Cys
Cys
UGU
UGC
AGU
AGC
Ser
Ser
AGA
AGG
Arg
Arg
*AUG signals translation initiation as well as coding for Met
3rd base irrelevant
Purines
Pyrimidines
3 out of 4
Unique definition
Unique definition

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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

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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
of anticodon
Base at 3' end
of codon
I*
G
U
A
C
A, C, or U
C or U
A or G
U
G
I* = hypoxanthine

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Wobble Base Pairing
N
N
O
H
N
N
R
N N
H
O
R
O
Inosine
Uridine
N N
N
R
O
Cytidine
N
N
O
H
N
N
R
Inosine
H
H
N
N
O
H
N
N
R
Inosine
N N
H
N
H
N
N
R
Adenosine

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Wobble Base Pairing
• The Wobble Hypothesis (proposed by Francis
Crick) 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

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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

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D Arm - Contains
dihydrouridine
Acceptor Arm - A specific
amino acid is attached to
the 3’ end (CCA-3’) TyC arm - y stands
for pseudouridine
Extra Arm - May
vary in size
Anticodon
Transfer RNA (tRNA)
U*
9
26
2223Pu
16
12Py 10
25
20:1
G*
17:1
Pu
A
20:2
17
13
20
G
A
5051
656463
G
62
52
C
Pu
59
y
A*
C
Py
T
49
39
41
42
31
29
28
Pu*
43
1
27
U
35
38
36
Py*
34
40
30 47:1
47:15
46
Py
47:16
45
44
47
73
C
C
A
70
71
72
66
67
68
69
3
2
1
7
6
5
4
Amino Acid attachment site
5’

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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

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Gly
Amino-
acyl-tRNA
Synthetase
Gly
Amino-
acyl-tRNA
Synthetase
P
Making
Aminoacyl-
tRNA
P
P
Pyrophosphate
P
P
P
ATP
Amino-
acyl-tRNA
Synthetase
P
Gly
CCA

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Amino-
acyl-tRNA
Synthetase
Making
Aminoacyl-
tRNA
Gly
CCA
Aminoacyl-
tRNA
Note that the amino acid is not paired with the
tRNA on the basis of the anticodon. The correct
tRNA for a given amino acid is recognized on
the basis of other parts of
the molecule.
©1998 Timothy G. Standish
Gly
P
P
Pyrophosphate
P
P
P
ATP
Amino-
acyl-tRNA
Synthetase
Gly
Amino-
acyl-tRNA
Synthetase
P
P
AMP
Amino-
acyl-tRNA
Synthetase
Gly
CCA

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Aminoacylation of tRNA
H
H
C
O
C
N
C
O
N
C
C
C
C
C
H
H
O
H
H
O
P
O
H
O
N
N
C
C
O
H H
H
N
H
H
3’
5’
H C
N
C
O
H
R H
H
O
H
O H

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Amino
acid
tRNA
Aminoacylation
of tRNA
3’
5’
H
H
C
O
C
N
C
O
N
C
C
C
C
C
H
H
O
H
H
O
P
O
H
O
N
N
C
C
O
H
H
N
H
H
H C
N
C
O
H
R H
H
O H
Class I Aminoacyl
tRNA Synthetases
attach amino acids to
the 2’ carbon while
Class II attach to
the 3’carbon

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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

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Amino Acid Activation
• Requires
• amino acids
• tRNAs
• aminoacyl-tRNA synthetases
• ATP, Mg2+
• Formation on an aminoacyl-tRNA
amino acid + aminoacyl-AMP + PPi
aminoacyl-AMP + tRNA aminoacyl-tRNA + AMP
amino acid + ATP + tRNA
aminoacyl-tRNA + AMP + PPi
ATP

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Amino Acid Activation
PPi
A-rib- O- P- OPOPO-
O-
O
O-
ATP
+ -
O- C- CH- R
O
NH3
+
Amino acid
A-rib- O- P- O- C-CH- R
O
NH3
+
An aminoacyl-AMP
Step 1:
O
O-
O
O-
O
AMP
H
A
H
OH HO
H H
O
t RNA
+
Transfer RNA
H
A
H
O HO
H H
O
t RNA
C-CH- R
NH3
+
O
An aminoacyl-tRNA
Step 2:
A-rib- O- P- O- C-CH- R
O
NH3
+
An aminoacyl-AMP
O-
O
PPi
A-rib- O- P- OPOPO-
O-
O
O-
ATP
+ -
O- C- CH- R
O
NH3
+
Amino acid
A-rib- O- P- O- C-CH- R
O
NH3
+
An aminoacyl-AMP
Step 1:
O
O-
O
O-
O
AMP
H
A
H
OH HO
H H
O
t RNA
+
Transfer RNA
H
A
H
O HO
H H
O
t RNA
C-CH- R
NH3
+
O
An aminoacyl-tRNA
Step 2:
A-rib- O- P- O- C-CH- R
O
NH3
+
An aminoacyl-AMP
O-
O

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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.

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Amino Acid Activation
Ribbon diagram of tRNA tertiary structure

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Chain Initiation (Prokaryotes)
• 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+

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Chain Initiation
• In Prokaryotes, the initial N-terminal amino acid
is N-formylmethionine (fmet)
Met + tRNAfmet
Met-tRNA
synthetase
(ATP)
Met-tRNAfmet
Formyl- FH4
FH4
Met-tRNAfmet
formyltransferase
CH3 -S-CH2 CH2 CHC- t RNA
O
NH
C
H O
N-Formylmethionine-tRNA fmet

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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

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Chain Initiation
• 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
(Australian scientists John Shine and Lyn 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

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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

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Chain Initiation
Formation of an initiation complex

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

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Chain Elongation
Sine-Delgano sequences recognition by E. coli

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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 next aminoacyl-tRNA occupies the empty A site

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Chain Elongation
H
H
O HO
H H
O
t RNA
An aminoacyl-tRNA
C= O
H2 N-CH
R
H
H
O HO
H H
O
t RNAfmet
N-Formylmethionine-tRNA fmet
C= O
H- C-NH- CH
CH3 SCH2 CH2
O
H
H
OH HO
H H
O
t RNAfmet
H
H
O HO
H H
O
t RNA
C= O
H- C-NH- CH-C- NH- CH
CH3 SCH2 CH2
O
R
O
peptidyl transferase
+
Adenine
Adenine
Adenine Adenine

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Chain Elongation
• puromycin
H
H
O HO
H H
O
C-CH- R
NH3
+
O
An aminoacyl-tRNA
N
N N
N
NH2
t RNA-OPO- CH2
O-
H
H
NH HO
H H
O
C-CH- CH2
NH3
+
O
N
N N
N
N
HO- CH2
CH3
H3 C
OCH3
Puromycin
O

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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

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Protein Synthesis
• Summary of required components
Step Components
Amino acid
activation
Chain initiation
Chain elongation
Chain termination
Amino acids, tRNAs, ATP, Mg 2+
,
fmet-tRNAfmet
, initiation codon (AUG),
30S and 50S ribosomal subunits, GTP, Mg 2+
,
and initiation factors (IF-1, IF-2, and IF-3)
70S ribosome, codons for mRNA, GTP, Mg 2+
,
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 Mg2+
and aminoacyl-tRNA synthetases

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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

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Protein Synthesis
Peptide chain termination

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Protein Synthesis
Simultaneous protein synthesis on polysomes

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Translation - Eukaryotes
Structure of eukaryotic mRNAs

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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

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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

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Polypeptide Modification
• Newly synthesized polypeptides are frequently
modified before they reach their final form
• 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 covalent modifications; e.g., addition of
carbohydrates

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9/21/2021
RAMON S. DEL FIERRO,Ph.D. (Tokyo)
Professor of Biochemistry