Chapter 13
Lecture Outline
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INTRODUCTION
n
n
The translation of the mRNA codons into amino
acid sequences leads to the synthesis of proteins
A variety of cellular components play important
roles in translation
n
n
These include proteins, RNAs and small molecules
In this chapter we will discuss the current state of
knowledge regarding the molecular features of
mRNA translation
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13-2
13.1 THE GENETIC BASIS FOR
PROTEIN SYNTHESIS
n
n
Proteins are the active participants in cell
structure and function
Genes that encode polypeptides are termed
structural genes
n
n
These are transcribed into messenger RNA (mRNA)
The main function of the genetic material is to
encode the production of cellular proteins
n
In the correct cell, at the proper time, and in suitable
amounts
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13-3
Beadle and Tatumʼs Experiments
n
In the early 1940s, George Beadle and Edward
Tatum were also interested in the relationship
between genes, enzymes and traits
n
They specifically asked this question
n
n
Is it One gene–one enzyme or one gene–many enzymes?
Their genetic model was Neurospora crassa (a
common bread mold)
n
Their studies involved the analysis of simple nutritional
requirements
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13-7
Beadle and Tatumʼs Experiments
n
n
n
They analyzed more than 2,000 strains that had
been irradiated to produce mutations
They analyzed enzyme pathways for synthesis of
vitamins and amino acids
Figure 13.2 shows an example of their findings on
the synthesis of the amino acid methionine
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13-8
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Neurospora!
growth"
1"
WT"
WT"
1"
24
4"
3"
Minimal"
WT"
1"
24
3"
WT"
1"
24
3"
+Cystathionine"
+O–acetylhomoserine"
WT"
1"
2 4
3"
+Homocysteine"
2"
3"
+Methionine"
(a) Growth of strains on minimal and supplemented growth media!
Homoserine"
O–acetylhomoserine"
Enzyme 1"
Enzyme 2"
Cystathionine"
Enzyme 3"
Homocysteine"
Methionine"
Enzyme 4"
(b) Simplified pathway for methionine biosynthesis!
Every mutant strain was blocked at a particular step in
the synthesis pathway, showing that each gene encoded
one enzyme
Figure 13.2
13-9
Beadle and Tatumʼs Experiments
n
In the normal strains, methionine was synthesized
by cellular enzymes
n
n
In the mutant strains, a genetic defect in one gene
prevented the synthesis of one protein required in one
step of the pathway to produce that amino acid
Beadle and Tatumʼs conclusion: A single gene
controlled the synthesis of a single enzyme
n
This was referred to as the one gene–one enzyme
hypothesis
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13-10
Beadle and Tatumʼs Experiments
n
In later decades, this theory had to be modified
n
n
1. Enzymes are only one category of proteins
2. Some proteins are composed of two or more different
polypeptides
n
n
n
n
The term polypeptide denotes structure
The term protein denotes function
So it is more accurate to say a structural gene encodes a
polypeptide
3. Many genes have been identified that do not encode
polypeptides
n
For instance, functional RNA molecules (tRNA, rRNA, etc.)
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13-11
The Genetic Code
n
Translation involves an interpretation of one
language into another
n
n
Translation relies on the genetic code
n
n
In genetics, the nucleotide language of mRNA is
translated into the amino acid language of proteins
Refer to Table 13.1
The genetic information is coded within mRNA in
groups of three nucleotides known as codons
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13-12
Three codons do not
encode an amino acid.
These are read as STOP
signals for translation
Triplet codons correspond
to a specific amino acid
Multiple codons may encode
the same amino acid.
These are known as
synonymous codons
13-13
n
Special codons:
n
AUG (which specifies methionine) = start codon
n
n
n
n
UAA, UAG and UGA = termination, or stop, codons
The code is degenerate
n
More than one codon can specify the same amino acid
n
n
For example: GGU, GGC, GGA and GGG all code for glycine
In most instances, the third base is the variable base
n
n
This defines the reading frame for all following codons
AUG specifies additional methionines within the coding sequence
It is sometime referred to as the wobble base
The code is nearly universal
n
Only a few rare exceptions have been noted
n
Refer to Table 13.3
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13-14
13-15
n
Figure 13.3 provides an overview of gene expression
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Coding strand
DNA
Transcription
5′
3′
5′
mRNA
3′
A C T G C C C A T G A G C G A C C A C T T G G G G C T C G G G G A A T A AC C G T C G A G G
T G A CG GG T A CT C G CT G G TG A A CC CC G A G CC CC T TA T TGGC AGC T C C
5′
Template strand
A C UG C C C A UG A G C G AC C A CU UG G G G C U CG G G G A A UA A C C G UC G A G G
5′ − untranslated"Start"
region
codon
Codons
3′
Stop" 3′ − untranslated"
codon
region
Anticodons
Translation
UAC UCG CUG GUG A AC CCC GAG CCC CUU
Polypeptide
tRNA
5′
3′
Figure 13.3
Met
Ser
Asp
His
Leu
Gly
Leu
Gly
Note that the start codon sets the
reading frame for all remaining
codons
Glu
13-16
Evidence that the Genetic Code is
Read in Triplets
n
n
The first such evidence came in 1961 from studies of Francis
Crick and his colleagues
These studies involved the isolation of phage T4 mutants
n
n
n
n
rII mutants produced large plaques with clear boundary
r+ (wild-type) produced smaller, fuzzy plaques
Crick et al exposed r+ phages to the chemical proflavin that causes
single-nucleotide additions or deletions
n rII mutant phages were recovered and analyzed
These mutants were then re-exposed to proflavin
+
n r phages were recovered and analyzed
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13-17
Evidence that the Genetic Code is
Read in Triplets
n
n
Different + or - mutations were introduced into the same
phage via crossing over, as described in Chapter 7
As shown in the hypothetical example of Table 13.2, the
wild-type plaque morphology is restored by
n
1. A (+) and a (-) mutation that are close to each other
n
n
2. Three (-)(-)(-) mutation combinations
n
n
AND MORE IMPORTANTLY
One or Two frameshifts combined produced mutants
These results are consistent with the idea that the genetic
code is read in multiples of three nucleotides
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13-18
13-19
Experiment 13A: Synthetic RNA
Helped Decipher the Genetic Code
n
The genetic code was deciphered in the early 1960s
n
n
Thanks to several research groups, including two headed by
Marshall Nirenberg and H. Gobind Khorana
Nirenberg and his colleagues used a cell-free translation
system that was developed earlier by other groups
n However, they made a major advance
n
n
They discovered that addition of synthetic RNA to DNase-treated
extracts restores polypeptide synthesis
Moreover, they added radiolabeled amino acids to these
extracts
n
Thus, the polypeptides would be radiolabeled and easy to detect
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13-20
n
To make synthetic RNA, the enzyme polynucleotide
phosphorylase was used
n
n
In the presence of excess ribonucleoside diphosphates (NDPs), it
catalyzes the covalent linkage of ribonucleotides into RNA
n Since it does not use a template, the order of nucleotides is
random
An experimenter can control the amounts of nucleotides added
n
For example, if 70% G and 30% U are mixed together, then …
Codon Possibilities
Percentage in the Random Polymer
GGG
0.7 x 0.7 x 0.7 = 0.34 = 34%
GGU
0.7 x 0.7 x 0.3 = 0.15 = 15%
GUU
0.7 x 0.3 x 0.3 = 0.06 = 6%
UUU
0.3 x 0.3 x 0.3 = 0.03 = 3%
UGG
0.3 x 0.7 x 0.7 = 0.15 = 15%
UUG
0.3 x 0.3 x 0.7 = 0.06 = 6%
UGU
0.3 x 0.7 x 0.3 = 0.06 = 6%
GUG
0.7 x 0.3 x 0.7 = 0.15 = 15%
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= 100%
13-21
The Hypothesis
n
The sequence of bases in RNA determines the
incorporation of specific amino acids into the
polypeptide
n
The experiment aims to help decipher the relationship
between base composition and particular amino acids
Testing the Hypothesis
n
Refer to Figure 13.4
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13-22
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Conceptual level
Experimental level
1. Add the cell-free translation system"
to each of 20 tubes.
2. To each tube, add random mRNA"
polymers of G and U made via"
polynucleotide phosphorylase using"
70% G and 30% U.
3. Add a different radiolabeled amino acid"
to each tube, and add the other 19"
non-radiolabeled amino acids. The"
translation system contained enzymes"
(discussed later) that attach amino acids"
to the appropriate tRNAs.
Cell-free"
translation system
For each"
tube:
Solution"
of G–U"
polymer
G G G
5ʼ
3ʼ
mRNA polymer
One radiolabeled"
amino acid"
(e.g., glycine)
19 other"
amino acids
G G G
5ʼ
4. Incubate for 60 minutes to allow"
translation to occur.
U G U G U G G
37°C
U G U G U G G
Polypeptide
Gly
3ʼ
Translation
Cys
Val
Radiolabeled amino acid
TCA
5. Add 15% trichloroacetic acid (TCA),"
which precipitates polypeptides but"
not amino acids.
Precipitated"
polypeptides
Water
6. Place the precipitate onto a filter and"
wash to remove unused amino acids.
7. Count the radioactivity on the filter in a"
scintillation counter (see the Appendix"
for a description).
8. Calculate the amount of radiolabeled"
amino acids in the precipitated"
polypeptides.
Figure 13.4
Precipitated"
polypeptides
Filter
Polypeptides
Scintillation"
counter
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13-23
The Data
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13-24
Interpreting the Data
Radiolabeled Amino
Acid Added
n
Relative Amount of
Radiolabeled Amino
Acid Incorporated
into Translated
Polypeptide (% of
total)
Glycine
49
Valine
21
Tryptophan
15
Cysteine
6
Leucine
6
Phenylalanine
3
The other 14 amino acids
0
Due to two codons:
GGG (34%) and GGU (15%)
Each is specified by a
codon that has one guanine
and two uracils (G + 2U)
But the particular sequence
for each of these amino
acids cannot be
distinguished
Consistent with the results of
an earlier experiment:
A random polymer with only
uracils encoded phenylalanine
It is important to note that the genetic code could not be deciphered in a
single experiment, but required combining data from multiple experiments
such as the one described here.
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13-25
RNA Copolymers Helped to Crack
the Genetic Code
n
In the 1960s, Gobind Khorana and his collaborators
developed a novel method to synthesize RNA
n
n
n
They first created short RNAs (2 to 4 nucleotides long) that
had a defined sequence
These were then linked together enzymatically to create
long copolymers
They used these copolymers in a cell-free translation
system like the one described in Figure 13.4
n
n
This was an important tool in identifying the codons
Refer to Figure 13.5 and Table 13.4
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13-26
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Proline
Proline tRNA
CCC
Triplet RNA that"
specifies proline
5′ – C C C – 3′
Ribosome
Filter
CCC
Ribosomes and tRNAs were mixed with a
synthesized triplet RNA encoding CCC. Only
tRNAs carrying proline bound the ribosome,
showing that CCC encodes proline
Unbound"
tRNA
Figure 13.5
13-27
13-28
A Polypeptide Chain Has Directionality
n
n
n
Polypeptide synthesis has a directionality that
parallels the 5ʼ to 3ʼ orientation of mRNA
During each cycle of elongation, a peptide bond is
formed between the carboxyl group of the last amino
acid in the polypeptide chain and the amino group in
the amino acid being added
The first amino acid has an exposed amino group
n
n
The last amino acid has an exposed carboxyl group
n
n
Said to be N-terminal or amino terminal end
Said to be C-terminal or carboxy terminal end
Refer to Figure 13.6
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13-29
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R1 O
H 3N +
C
C
H
R2 O
N
C
H
H
R1 O
H 3N +
C
C
H
R3 O
C
C
H
H
+ H3N+" C
N
C
C
H
H
O–
R2 O
N
R4 O
H
R3 O
C
N
C
H
H
C
C
O–
R4 O
N
C
C + H 2O
H
H
O–
Last peptide bond formed in the"
growing chain of amino acids
(a) Attachment of an amino acid to a peptide chain
OH
CH3
S
CH2
OH
CH2
CH2
H 3C
H
+
Amino" H3N
terminal"
end
C
C
H
O
Methionine
N
H
C
C
H
O
Serine
N
SH
CH3
CH
CH2
H
C
C
H
O
Valine
N
CH2
H
C
C
H
O
Tyrosine
N
C
C
H
O
O– Carboxyl"
terminal"
end
Cysteine
Peptide bonds
5′
AUG
AGC
GU U
UAC
UGC
3′
Sequence in mRNA
Figure 13.6
(b) Directionality in a polypeptide and mRNA
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13-30
n
There are 20 amino acids that may be found in polypeptides
n Each contains a different side chain, or R group
n Each R group has its own particular chemical properties
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CH3
H
H 3N
CH3 CH3
CH
CH3
+
COO–
C
+
H 3N
H
Glycine (Gly) G
C
COO–
+
H 3N
H
Alanine (Ala) A
C
CH3
CH3 CH3
CH
CH2
COO–
H
Valine (Val) V
+
H 3N
C
CH2
CH3
COO–
H
Leucine (Leu) L
S
+
H 3N
CH
C
COO–
H
Isoleucine (Ile) I
+
H 2N
C
COO–
H
Proline (Pro) P
CH2
SH
CH2
CH2 CH2
CH2
CH2
+
H 3N
C
COO–
+
H 3N
C
COO–
H
H
Cysteine (Cys) C Methionine (Met) M
(a) Nonpolar, aliphatic amino acids
H
OH
n
N
CH2
+
H 3N
C
CH2
COO–
+
H 3N
C
n
CH2
COO–
H
H
Phenylalanine (Phe) F Tyrosine (Tyr) Y
+
H 3N
C
Nonpolar amino acids are
hydrophobic
COO–
H
Tryptophan (Trp) W
They are often buried
within the interior of a
folded protein
(b) Aromatic amino acids
Figure 13.7
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13-31
n
Polar and charged amino acids are hydrophilic
n
They are more likely to be on the surface of a protein
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O
O
HCOH
CH2
+
H 3N
COO–
C
H
Serine (Ser) S
+
H 3N
CH2
CH2
COO–
C
NH2
C
CH3
OH
H
Threonine (Thr) T
+
H 3N
C
NH2
C
CH2
COO–
+
H 3N
C
COO–
H
H
Asparagine (Asn) N Glutamine (Gln) Q
(c) Polar, neutral amino acids
NH2
+
O–
O
C
O–
O
C
H 3N
C
+
NH
CH2
CH2
+
HN
CH2
COO–
+
H 3N
C
CH2
COO–
H
H
Aspartic acid (Asp) D Glutamic acid (Glu) E
(d) Polar, acidic amino acids
+
H 3N
C
C
CH2
NH
CH2
CH2
NH
CH2
CH2
CH2
CH2
COO–
H
Histidine (His) H
+
H 3N
N
+
NH3
C
CH3
+
H 3N
C
C
O
CH2
CH2
COO–
H
Lysine (Lys) K
(e) Polar, basic amino acids
NH2
COO–
H
Arginine (Arg) R
CH2
SeH
CH2
+
H 3N
C
CH2
COO–
+
H 3N
C
COO–
H
H
Selenocysteine (Sec) Pyrrolysine (Pyl)
(f) Nonstandard amino acids
Figure 13.7
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13-32
Levels of Structure in Proteins
n
There are four levels of structure in proteins
n
n
n
n
n
1.
2.
3.
4.
Primary
Secondary
Tertiary
Quaternary
A proteinʼs primary structure is its amino acid
sequence
n
Refer to Figure 13.8
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13-33
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Arg Cys Glu
Phe Gly
Leu
1
Val
Lys
n
10
Ala
Ala
Ala
NH3+
Met
Lys
20
Leu
Gly Arg
His
Tyr Asn
Asp Leu Gly
Tyr
Ser
Arg
Gly
30
Asn
The amino acid
sequence of the
enzyme
lysozyme
Val Cys Ala Ala
Trp
Lys Phe Glu
Ser
Asn
Phe
Asn Arg Asn
Thr
Thr
Ala
Asp
40 Asn
Within the cell, the
protein will not be
found in this linear
state
n Rather, it will adopt
a compact 3-D
structure
Gin Thr
Gly
50
Ser
60
Thr
Asp
Tyr Gly
lle
Leu
Gln
Asn
lle
Ser
Cys
Asn
70
Leu Asn Arg Ser
Gly Pro Thr
Cys
n
Arg Trp Trp
Asp
Gly
Arg
Indeed, this folding
can begin during
translation
Asn
129 amino acids
long
lle
Pro
80
Cys
Gly
Leu
Ser Ala
Leu
Ser
Ser
Asp
Gly
Asp Ser
Val
Thr
Ser
lle Lys Lys Ala Cys
Asn
100
Asn
Ala Trp
Val Ala Trp
110
Arg Asn
Arg Cys
Lys
129
Gly
Leu
Arg
COO–
Cys Gly Arg
lle
90
Ala
Asp
Met
Figure 13.8
lle
Trp Ala Gln
120
Val
Thr
Val
n
The progression from
the primary structure to
the 3-D structure is
dictated by the amino
acid sequence within
the polypeptide
Asp
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13-34
Levels of Structures in Proteins
n
n
The primary structure of a protein folds to form
regular, repeating shapes known as secondary
structures
There are two types of secondary structures
n
α helix
β sheet
n
Certain amino acids are good candidates for each structure
n
n
n
These secondary structures are stabilized by the
formation of hydrogen bonds between atoms located in
the polypeptide backbone
Refer to Figure 13.9
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13-35
Levels of Structures in Proteins
n
The short regions of secondary structure in a protein
fold into a three-dimensional tertiary structure
n
n
n
n
Refer to Figure 13.9
This is the final conformation of proteins that are
composed of a single polypeptide
Structure determined by hydrophobic and ionic interactions as well as
hydrogen bonds and Van der Waals interactions
Proteins made up of two or more polypeptides have
a quaternary structure
n
n
This is formed when the various polypeptides associate
with one another to make a functional protein
Refer to Figure 13.9
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13-36
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Primary!
structure
Tertiary!
structure
Secondary!
structure
Quaternary!
structure
C
Phe
Glu
O
N
C
C
O
H
H
N
C
C
C
H
Leu
N
Iso
N
C
HO
O N
Tyr
Regions of"
NH3+
secondary"
structure and"
irregularly shaped"
regions fold into a"
–
three-dimensional" COO
conformation.
N
C
C
O
H
C
C
H
C
H
COO–
(c)
C
C
N
NH3+
N
O
C
Two or more"
polypeptides"
may associate"
with each other.
Protein"
subunit
O
O
(d)
α helix
Ala
(a)
H
NH3+
Val
C
Depending on"
the amino acid"
sequence,"
some regions"
may fold into"
an α helix or"
β sheet.
COO–
Ala
O
C
CN C
H
H
O
CCNCC
O
H
H
O
CC N CC
O H
H
O
CC N CC
O H
H
C
C
O
C H O
C H O
C N C
C H O
N
C
C
C
H O C
C H
C N C
H O C
C N C
H O C
H O C
β sheet
(b)
O
Figure 13.9
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13-37
Functions of Proteins
n
n
n
To a great extent, the characteristics of a cell depend on the
types of proteins its makes
Proteins can perform a variety of functions
n Refer to Table 13.5
A key category of proteins are enzymes
n
n
Accelerate chemical reactions within a cell
Can be divided into two main categories
n Anabolic enzymes à Synthesize molecules and macromolecules
n Catabolic enzymes à Break down large molecules into small ones
n
Important in generating cellular energy
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13-38
13-39
13.2 STRUCTURE AND
FUNCTION OF tRNA
n
In the 1950s, Francis Crick and Mahon Hoagland
proposed the adaptor hypothesis
n
n
tRNAs play a direct role in the recognition of codons in
the mRNA
In particular, the hypothesis proposed that tRNA
has two functions
n
n
1. Recognizing a 3-base codon in mRNA
2. Carrying an amino acid that is specific for that codon
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13-40
Recognition Between tRNA and mRNA
n
During mRNA-tRNA recognition, the anticodon in
tRNA binds to a complementary codon in mRNA
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Phenylalanine
Proline
tRNAs are named
according to the
amino acid they bear
The anticodon is
anti-parallel to
the codon
tRNAPhe
tRNAPro
A A G
G GC
Phenylalanine"
anticodon
Proline"
anticodon
U UC
C C G
5′
Figure 13.10
3′ mRNA
Phenylalanine"
codon
Proline"
codon
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13-41
tRNAs Share Common Structural
Features
n
The secondary structure of tRNAs exhibits a
cloverleaf pattern
n
It contains
n
n
n
n
n
Three stem-loop structures
A few variable sites
An acceptor stem with a 3ʼ single strand region
The actual three-dimensional or tertiary structure
involves additional folding
In addition to the normal A, U, G and C nucleotides,
tRNAs commonly contain modified nucleotides
n
More than 80 of these can occur
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13-49
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NH3+
H C R
C O
3′
A
C
C
Found in all tRNAs
OH
O
A
C
C
Acceptor stem
5′
Covalent"
bond"
between"
tRNA"
and an"
amino"
acid
PO4
70
Stem–loop
60
U
UH2 G
C
A
U
10
G
m2 G
UH2
A
G UH2
19
40
30
U
P
U
mI
I
A
G
50
Figure 13.12 Structure of tRNA
U
G
C
T
P
Not found in all tRNAs
Other variable sites are
shown in blue as well
C
The modified bases are:
n
I = inosine
n
mI = methylinosine
n
T = ribothymidine
n
UH2 = dihydrouridine
n
m2G = dimethylguanosine
n
ψ = pseudouridine
Anticodon
13-50
Charging of tRNAs
n
The enzymes that attach amino acids to tRNAs are
known as aminoacyl-tRNA synthetases
n
There are 20 types
n
n
One for each amino acid
Aminoacyl-tRNA synthetases catalyze a two-step
reaction involving three different molecules
n
Amino acid, tRNA and ATP
n
Refer to Figure 13.13
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13-51
Charging of tRNAs
n
The aminoacyl-tRNA synthetases are responsible
for the “second genetic code”
n
n
n
n
The selection of the correct amino acid must be highly
accurate or the polypeptides may be nonfunctional
Error rate is less than one in every 100,000
Sequences throughout the tRNA including but not limited
to the anticodon are used as recognition sites
Modified bases may affect
n translation rates
n recognition by aminoacyl-tRNA synthetases
n Codon-anticodon recognition
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13-52
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Aminoacyl-tRNA"
synthetase
Specific"
amino acid
P
P
P
A
ATP
An amino acid and ATP bind to"
the enzyme. AMP is covalently"
bound to the amino acid, and"
pyrophosphate is released.
P
A
P P
Pyrophosphate
The correct tRNA binds to the"
enzyme. The amino acid"
becomes covalently attached to"
the 3′ end of the tRNA. AMP is"
released.
tRNA
3′
5′
3′
5′
P
A
The amino acid is
attached to the 3ʼ end
of the tRNA by an
ester bond
AMP
The “charged” tRNA is"
released.
5′
Figure 13.13
3′
13-53
tRNAs and the Wobble Rule
n
As mentioned earlier, the genetic code is degenerate
n
n
With the exception of serine, arginine and leucine, this
degeneracy always occurs at the codonʼs third position
To explain this pattern of degeneracy, Francis Crick
proposed in 1966 the wobble hypothesis
n
n
In the codon-anticodon recognition process, the first two
positions pair strictly according to the A – U /G – C rule
However, the third position can actually “wobble” or move
a bit
n
Thus tolerating certain types of mismatches
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13-54
Phenylalanine
tRNAs that can recognize the same
codon are termed isoacceptor tRNAs
5′
3′
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Nucleotide of"
Third nucleotide"
of tRNA anticodon" of mRNA codon
A A G
U U U
Wobble"
position
n
3′
5′
G
C
A
U
inosine I
5-methyl-2-thiouridine xm5s2U"
n
5-methyl-2ʼ-O-methyluridine xm5Um
n
2ʼ-O-methyluridine Um
position
n
5-methyluridine xm5U
C, U
G
U, C, G, (A)
A, U, G, (C)
U, C, A
n
(a) Location of wobble
n
5-hydroxyuridine xo5U"
n
lysidine k2C
A, (G)
U, A, G
A
(b) Revised wobble rules
Figure 13.14 Wobble position and base pairing rules
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
Recognized
very poorly by
the tRNA
13-55
13.3 RIBOSOME STRUCTURE
AND ASSEMBLY
n
n
Translation occurs on the surface of a large
macromolecular complex termed the ribosome
Bacterial cells have one type of ribosome
n
n
Found in their cytoplasm
Eukaryotic cells have two types of ribosomes
n
n
One type is found in the cytoplasm
The other is found in organelles
n
Mitochondria ; Chloroplasts
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13-56
13.3 RIBOSOME STRUCTURE
AND ASSEMBLY
n
n
Unless otherwise noted the term eukaryotic
ribosome refers to the ribosomes in the cytosol
A ribosome is composed of structures called the
large and small subunits
n
Each subunit is formed from the assembly of
n
n
n
Proteins
rRNA
Table 13.6 presents the composition of bacterial and
eukaryotic ribosomes
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13-57
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13-58
Functional Sites of Ribosomes
n
During bacterial translation, the mRNA lies on the
surface of the 30S subunit
n
n
Ribosomes contain three discrete sites
n
n
n
n
As a polypeptide is being synthesized, it exits through a
channel within the 50S subunit
Peptidyl site (P site)
Aminoacyl site (A site)
Exit site (E site)
Ribosomal structure is shown in Figure 13.15
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13-59
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Polypeptide
tRNA
E
P
A
50S
30S
mRNA
5ʹ′
3ʹ′
(c) Model for ribosome structure
Figure 13.15
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13-60
13.4 STAGES OF
TRANSLATION
n
Translation can be viewed as occurring in three
stages
n
n
n
n
Initiation
Elongation
Termination
Refer to 13.16 for an overview of translation
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13-61
Initiator tRNA
aa1
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
aa1
Initiator"
tRNA – tRNA"
with first"
amino acid
Large
E
Ribosomal"
subunits
UAC"
Anticodon
Small
A
AUG"
Start codon
mRNA
UAG"
Stop codon
5′
P
Initiation
3′
5′
AUG"
Start codon
3′
Elongation!
(This step"
occurs many"
times.)
aa1
aa2
aa3
aa4
Recycling of translational"
components
Release"
factor
Completed"
polypeptide
E
P
aa5
E
A
P
A
Termination
UAG"
Stop codon
5′
Figure 13.16
3′
5′
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
3′
13-62
The Translation Initiation Stage
n
The mRNA, initiator tRNA, and ribosomal subunits
associate to form an initiation complex
n
n
This process requires three Initiation Factors
The initiator tRNA recognizes the start codon in
mRNA
n
In bacteria, this tRNA is designated tRNAfmet
n
n
It carries a methionine that has been covalently modified to
N-formylmethionine
The start codon is AUG, but in some cases GUG or UUG
n
In all three cases, the first amino acid is N-formylmethionine
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13-63
n
The binding of mRNA to the 30S subunit is facilitated by a
ribosomal-binding site or Shine-Dalgarno sequence
n
This is complementary to a sequence in the 16S rRNA
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Component of the
30S subunit
3′
5′
AG
16S rRNA
Hydrogen bonding
mRNA
A UCU AGU A AGGAGGUUGU A UGGUU C AGCGC A CG
Figure 13.18
n
A UUCC UC C A C
Shine-Dalgarno"
sequence
CAG
3′
Start"
codon
Figure 13.17 outlines the steps that occur during
translational initiation in bacteria
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13-64
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
IF1 and IF3 bind to the 30S subunit.
IF3
The mRNA binds to the 30S subunit."
The Shine-Dalgarno sequence is"
complementary to a portion of the"
16S rRNA.
Portion of"
16S rRNA
IF3
5′
30S subunit
IF1
IF1
Start"
Shine-"
codon
Dalgarno"
sequence"
(actually 9"
nucleotides long)
3′
IF2, which uses GTP, promotes"
the binding of the initiator tRNA"
to the start codon in the P site.
Figure 13.17
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13-65
tRNAfMet
Initiator tRNA
GTP
IF2
IF1
IF3
3′
5′
IF1 and IF3 are released.
IF2 hydrolyzes its GTP and is released.
The 50S subunit associates.
tRNAfMet
70S initiation
complex
E
Figure 13.17
5′
P
A
70S"
initiation"
complex
This marks the
end of the
initiation stage
3′
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13-66
The Translation Initiation Stage
n
In eukaryotes, the assembly of the initiation complex
is similar to that in bacteria
n
However, additional factors are required
n
n
n
Note that eukaryotic Initiation Factors are denoted eIF
Refer to Table 13.7
The initiator tRNA is designated tRNAmet
n
It carries a methionine rather than a formylmethionine
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13-67
n
The start codon for eukaryotic translation is AUG
n
n
Ribosome scans from the 5ʼ end of mRNA until it finds
the AUG start codon (not all AUGs can act as a start)
The consensus sequence for optimal start codon
recognition is show here
Most important positions for codon selection
n
n
C C A U G G
-2 -1 +1 +2 +3 +4
These rules are called Kozakʼs rules
n
n
G C C (A/G)
-6 -5 -4
-3
Start codon
After Marilyn Kozak who first proposed them
With that in mind, the start codon for eukaryotic
translation is usually the first AUG after the 5ʼ Cap!
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13-68
n
Translational initiation in eukaryotes can be
summarized as such:
n
n
n
n
n
n
An initiation factor protein complex (eIF4) binds to the 5ʼ
cap in mRNA
These are joined by a complex consisting of the 40S
subunit, tRNAmet, and other initiation factors
The entire assembly moves along the mRNA scanning
for the right start codon
Once it finds this AUG, the 40S subunit binds to it
The 60S subunit joins
This forms the 80S initiation complex
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13-69
The Translation Elongation Stage
n
n
n
During this stage, amino acids are added to the
polypeptide chain, one at a time
The addition of each amino acid occurs via a series
of steps outlined in Figure 13.19
This process, though complex, can occur at a
remarkable rate
n
n
In bacteria à 15-20 amino acids per second
In eukaryotes à 2-6 amino acids per second
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13-70
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aa1
aa2
Ribosome
aa3
E site
A site
P site
5′
Codon 4
Codon 3
aa1
aa2
aa3
3′
aa4
mRNA
E
A charged tRNA binds"
to the A site. EF-Tu"
facilitates tRNA binding"
and hydrolyzes GTP.
The 23S rRNA (a component of
the large subunit) is the actual
peptidyl transferase
Thus, the ribosome
is a ribozyme!
Figure 13.19
P
A
3′
5′
Peptidyltransferase, which"
is a component of the 50S"
subunit, catalyzes peptide"
bond formation between the"
polypeptide and the amino"
acid in the A site.The"
polypeptide is transferred"
to the A site.
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13-71
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
aa1
aa2
tRNAs at the P and A
sites move into the E
and P sites,
respectively
aa3
aa4
E
P
A
aa1
aa2
aa3
3′
5′
aa4
The ribosome translocates"
1 codon to the right. This"
translocation is promoted"
by EF-G, which hydrolyzes"
GTP.
aa3
aa4
aa2
E
P
A
Codon 3
aa1
Codon 5
5′
E
P
Codon 4
3′
An uncharged"
tRNA is released"
from the E site.
A
Codon 3
5′
Figure 13.19
Codon 5
Codon 4
3′
This process is repeated, again and"
again, until a stop codon is reached.
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13-72
The Translation Elongation Stage
n
16S rRNA (a part of the 30S ribosomal subunit) plays
a key role in codon-anticodon recognition
n
It can detect an incorrect tRNA bound at the A site
n
n
It will prevent elongation until the mispaired tRNA is released
This phenomenon is termed the decoding function of
the ribosome
n
It is important in maintaining the high fidelity of mRNA
translation
n
Error rate: 1 mistake per 10,000 amino acids added
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13-73
The Translation Termination Stage
n
The final stage occurs when a stop codon is
reached in the mRNA
n
In most species there are three stop or nonsense codons
n
n
n
n
UAG
UAA
UGA
These codons are not recognized by tRNAs, but by
proteins called release factors
n
Indeed, the 3-D structure of release factors mimics that of tRNAs
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13-74
The Translation Termination Stage
n
Bacteria have three release factors
n
n
n
RF1, which recognizes UAA and UAG
RF2, which recognizes UAA and UGA
RF3, which does not recognize any of the three codons
n
n
It binds GTP and helps facilitate the termination process
Eukaryotes only have one release factor
n
eRF, which recognizes all three stop codons
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13-75
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tRNA in P"
site carries"
completed"
polypeptide
E
5′
P
A
Stop codon"
in A site
3′
mRNA
A release factor (RF) binds to the A site.
E
P
A
Release"
factor
3′
5′
The polypeptide is cleaved from the tRNA"
in the P site. The tRNA is then released.
3′
5′
The ribosomal subunits, mRNA, and"
release factor dissociate.
+
50S subunit
Figure 13.20
mRNA
30S subunit
3′
5′
13-76
13-77
Bacterial Translation Can Begin
Before Transcription Is Completed
n
Bacteria lack a nucleus
n
n
As soon an mRNA strand is long enough, a ribosome will
attach to its 5ʼ end
n
n
n
n
Therefore, both transcription and translation occur in the cytoplasm
So translation begins before transcription ends
This phenomenon is termed coupling
Refer to Figure 13.21
A polyribosome or polysome is an mRNA transcript that has
many bound ribosomes in the act of translation
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13-78
Coupling between transcription and translation in bacteria
Figure 13.21
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13-79
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13-80