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Chapter 13
TRANSLATION OF mRNA
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13.1 THE GENETIC BASIS FOR PROTEIN
SYNTHESIS
The translation of the mRNA codons into aminoacid sequences leads to the synthesis ofproteins
Proteins are the active participants in cell structureand function
Genes that encode polypeptides are termedstructural genes These are transcribed into messenger RNA (mRNA)
The main function of the genetic material is toencode the production of cellular proteins In the correct cell, at the proper time, and in suitable
amounts2
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In the 1940s, Beadle and Tatum were interested inthe relationship between genes, enzymes and traits
They specifically asked this question
Is it One geneone enzyme or one genemany enzymes?
Their genetic model was Neurospora crassa (a
common bread mold)
Their studies involved the analysis of simple nutritional
requirements
Beadle and Tatums Experiments
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They analyzed more than 2,000 strains that had
been irradiated to produce mutations
They analyzed enzyme pathways for synthesis ofvitamins and amino acids
Figure 13.2 shows an example of their findings onthe synthesis of the amino acidArginine
Beadle and Tatums Experiments
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Figure 13.2
Every mutant strain was blocked at a particular step in the
synthesis pathway, showing that each gene encodedone enzyme
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In the normal strains, arginine was synthesized by
cellular enzymes
In the mutant strains, a genetic defect in one gene
prevented the synthesis of one protein required in onestep of the pathway to produce that amino acid
Beadle and Tatums conclusion: A single gene
controlled the synthesis of a single enzyme This was referred to as the one geneone enzyme
hypothesis
Beadle and Tatums Experiments
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Translation involves an interpretation of onelanguage into another
In genetics, the nucleotide language of mRNA is
translated into the amino acid language of proteins
Translation relies on the genetic code
Refer to Table 13.1
The genetic information is coded within mRNA in
groups ofthree nucleotides known as codons
The Genetic Code
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Figure 13.3
Figure 13.3 provides an overview of gene expression
Note that the start codon sets the
reading frame for all remaining codons
5
Template strand
Coding strand
Transcription
3
Translation
DNA
mRNA
tRNAPolypeptide
5 untranslated
region
3untranslated
region
Start
codon
Codons Anticodons
3
3
5
5
A C T G C C C A T G G G G C TC G A CA G GC G G G A A T A A C C G T C G A G G
G G C A G C T C C
C C G U C G A G G
T T GC A C
T G A C G G G T A C C C C G AG C T GT C CG C C C T T A T TA A CG T G
5 3A C U G C C C A U G G G G C UC G A CA G GC G G G A A U A AU U GC A C
Met Gly LeuSer Asp Gly GluHis Leu
Stop
codon
UAC CCC GAGUCG CUG CCC CUUGUG A AC
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Multiple codons may encode
the same amino acid.
These are known assynonymous codons
Three codons do not
encode an amino acid.
These are read as STOP
signals for translation
A code of 3 nucleotides could code for a
maximum of43 or64 amino acids; there are
20 standard amino acids 10
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Specialcodons:
AUG (which specifies methionine) = start codon
This defines the reading frame for all following codons AUG specifies additional methionines within the coding sequence
UAA, UAG and UGA = termination, orstop, codons
The code is degenerate More than one codon can specify the same amino acid
For example: GGU, GGC, GGA and GGG all code forglycine
In most instances, the third base is the variable base
It is sometime referred to as the wobble base
The code is nearly universal
Only a few rare exceptions have been noted
Refer to Table 13.3 11
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Polypeptide synthesis has a directionality that
parallels the 5 to 3 orientation of mRNA
During protein synthesis, a peptide bond is formed
between the carboxyl group of the last amino acid in
the polypeptide chain and the amino group in theamino acid being added
The first amino acid has an exposed amino group
Said to be N-terminal or amino terminal end The last amino acid has an exposed carboxyl group
Said to be C-terminal or carboxy terminal end
Refer to Figure 13.6
A Polypeptide Chain Has Directionality
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Peptide bond formation
H 3 N+ O
H HO
H3C
Amino
terminalend
Carboxyl
terminalend
Methionine Serine
Peptide bonds
Sequence in mRNA
Valine
CH2
CH3
CH3
CH2
CH2
OH
CH
S
C C CN
H
O
C CN C
H O H
Cysteine
CH2
SH
CN
H
O
C
Tyrosine
CH2
OH
H
CN C
H O
5 3A U G A G C GU U U A C U G C
H
Figure 13.6 Directionality in a polypeptide and mRNA14
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Figure 13.7
There are 20 amino acids that may be found in polypeptides Each contains a different side chain, orR group Each R group has its own particular chemical properties
Nonpolar amino acids arehydrophobic They are often buried within the
interior of a folded protein
H
H
Glycine (Gly) G
(a) Nonpolar, aliphatic amino acids
H3N C COO
CH3 CH3
CH
H
Alanine (Ala) A
H3N COO
CH3 CH3
CH
CH2
H
Valine (Val) V
H3N C COO
+
CH2CH2
CH2
H
Proline (Pro) P
H2N C COO
+
CH2
CH3
CH3 CH
H
Leucine (Leu) L Methionine (Met) M
H3N C COO
+
Cysteine (Cys) C
+
CH2
SH
H
H3N C COO
CH2
CH2
CH3
S
H
H3N C COO
+
H
Isoleucine (Ile) I
H3N C COO
+
(b) Aromatic amino acids
Phenylalanine (Phe) F Tyrosine (Tyr) Y
H
H3N C COO
+CH2
H
H3N C COO
+CH2
OH
Tryptophan (Trp) W
H
H3N C COO
+CH2
N
H
+
CH3
C
+
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There are four levels of structure in proteins
1. Primary
2. Secondary
3. Tertiary 4. Quaternary
A proteins primary structure is its amino acid
sequence Refer to Figure 13.8
Levels of Structure in Proteins
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Lys
NH3+
110
20
30
40
50
60
70
80
90
100
110
120
129
ValPhe Gly
Arg Cys GluLeu
Ala
Ala
Ala
Met
Lys
Arg
His
GlyLeuAsp
AsnTyrArgGlyTyr
Ser
Thr
AspTyr
GlyLeu
Asn
SerGluPheLysAlaAlaCysValTrpAsn
LeuGly
Phe
Asn
ThrGinAla
ThrAsnArgAsn
Thr
Asp
Gly
Ser
lle
Glnlle
AsnSer
Arg Trp Trp
Cys
Asn
AspGly
ArgThrProGlySer
ArgAsnLeuCys
Asn
lle
Pro
CysSer Ala Leu
LeuSer
SerAsp
lleThr
Arg AsnArg
Cys
Lys
Gly
Thr
Asp
AlaTrp ValAla
Asn
Met
GlyAsp
GlyAsp Ser Val lle Lys Lys Ala
CysAsn
Val
Ser
Ala
ValGlnAlaTrplleArgGlyCys
Arg
Leu
Trp
COO
Figure 13.8
The amino acid
sequence of the
enzyme
lysozyme
129 amino acids
long
Within the cell, theprotein will not be foundin this linear state
Rather, it will adopt a
compact 3-Dstructure
Indeed, this foldingcan begin during
translation
The progression fromthe primary structure tothe 3-D structure is
dictated by the aminoacid sequence withinthe polypeptide
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Figure 13.9
helix
sheet
Primary
structure
Secondary
structure
Quaternary
structure
Tertiary
structure
Proteinsubunit
AlaC
O
C
C
C
C
O
O
Val
Phe
Glu
Tyr
Leu
Iso
Ala
H
N
NH3+
NH3+
COO
COO
NH3+
COO
H
N
CC
CC O
O
HH
NN
H
N
CC
C
CC
CO
OC
O
H
H
N
NN
Depending onthe amino acidsequence,some regionsmay fold intoan helix or sheet.
Two or morepolypeptidesmay associatewith each other.
Regions ofsecondarystructure andirregularly shapedregions fold into athree-dimensionalconformation.
(a)
(b)
(c)
(d)
H
Levels of Structures in Proteins
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The primary structure of a protein folds to formregular, repeating shapes known as secondary
structures
There are two types of secondary structures a helix
b sheet
Refer to Figure 13.9
Levels of Structures in Proteins
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The short regions of secondary structure in a protein
fold into a three-dimensional tertiary structure 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 oftwo or more polypeptides have
a quaternary structure
This is formed when the various polypeptides associate
with one another to make a functional protein
Levels of Structures in Proteins
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tRNA has two functions1. Recognizing a 3-base codon in mRNA
2. Carrying an amino acid that is specific for that codon
During mRNA-tRNA recognition, the anticodon in tRNA binds to a
complementary codon in mRNA
13.2 STRUCTURE AND FUNCTION OF tRNA
tRNAs are named
according to the
amino acid they bear
The anticodon isanti-parallel to
the codon
Phenylalanine
tRNAPhe tRNAPro
Phenylalanineanticodon
Phenylalaninecodon
Prolinecodon
A G
Proline
Prolineanticodon
U C
3 mRNA5
G CA G
U C C G
21
RNA Sh C S l F
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tRNAs Share Common Structural Features
The secondary structure of
tRNAs exhibits a cloverleafpattern; It contains 3 stem-loop structures
A few variable sites
An acceptor stem with a 3 singlestrand region
In addition to the normalnucleotides, tRNAs commonly
contain 80 modified nucleotides
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The enzymes that attach amino acids to tRNAs are
known as aminoacyl-tRNA synthetases
There are 20 types
One for each amino acid
Aminoacyl-tRNA synthetases catalyze a two-step
reaction involving three different molecules
Amino acid, tRNA and ATP
Refer to the figure next slide
Charging of tRNAs
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The amino acid is attached to the 3 end of the tRNA by an ester bond
Charging of tRNAs
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13.3 RIBOSOME STRUCTURE AND
ASSEMBLY
Translation occurs on the surface of the ribosome
Bacterial cells have one type of ribosome
Found in the cytoplasm
Eukaryotic cells have two types of ribosomes One type is found in the cytoplasm
The other is found in organelles
Mitochondria ; Chloroplasts
A ribosome is composed of structures called the large
and small subunits
Each subunit is formed from the assembly of
Proteins
rRNA
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13 3 RIBOSOME STRUCTURE AND
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13.3 RIBOSOME STRUCTURE AND
ASSEMBLY
A ribosome is composed of structures called the large and small subunits27
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During bacterial translation, the mRNA lies on the
surface of the 30S subunit As a polypeptide is being synthesized, it exits through a
channel within the 50S subunit
Ribosomes contain three discrete sites Peptidyl site (P site)
Aminoacyl site (A site)
Exit site (E site)
Functional Sites of Ribosomes
Model for ribosome structure
Polypeptide
30S
50S
35
tRNA
mRNA
E P A
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13 4 STAGES OF TRANSLATION
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mRNA
UACAnticodon
InitiatortRNA tRNA
with firstamino acid
AUGStart codon
AUGStart codon
UAGStop codon
UAGStop codon
Completedpolypeptide
3. Termination
2. Elongation(This stepoccurs manytimes.)
Recycling of translationalcomponents
Releasefactor
Small
LargeRibosomalsubunits
EEA
E AP
aa1aa2aa3aa4
aa5
aa1aa1
33 55
35
35
P P A
Figure 13.16
Initiator tRNA
1. Initiation
13.4 STAGES OF TRANSLATION
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The mRNA, initiator tRNA, and ribosomal subunits
associate to form an initiation complex
This process requires three Initiation Factors
The initiator tRNA recognizes the start codon in
mRNA
In bacteria, this tRNA is designated tRNAfmet
It carries a methionine that has been covalently modified toN-formyl-methionine
The start codon is AUG
The Translation Initiation Stage
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Shine-Dalgarno
sequence
mRNA
5 3A U C U A G U A A G U U C A GG G U CG A GU C A C G C A GU G GG U A
3
Start
codon
A U U C C C AC 16S rRNAU
The binding of mRNA to the 30S subunit is facilitated by a
ribosomal-binding site orShine-Dalgarno sequence
This is complementary to a sequence in the 16S rRNA
Figure 13.18
Hydrogen bonding
Component of the
30S subunit
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Figure 13.17
IF2, which uses GTP, promotesthe binding of the initiator tRNAto the start codon in the P site.
Portion of16S rRNA
The mRNA binds to the 30S subunit.The Shine-Dalgarno sequence iscomplementary to a portion of the16S rRNA.
IF1 and IF3 bind to the 30S subunit.
35
30S subunit
Shine-Dalgarnosequence
(9 nucleotideslong)
Startcodon
IF3 IF1
IF1IF3
32
tRNAfMet
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Figure 13.17
70S
initiation
complex
This marks
the end of the
initiation
stage
IF1 and IF3 are released.
IF2 hydrolyzes its GTP and is released.
The 50S subunit associates.
tRNAfMet
IF2GTP
E AP
35
35
70Sinitiationcomplex
IF1IF3
Initiator tRNA
tRNAfMet
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34
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In eukaryotes, the assembly of the initiation complex
is similar to that in bacteria
However, additional factors are required
Note that eukaryotic Initiation Factors are denoted eIF
Refer to Table 13.7
The initiator tRNA is designated tRNAmet It carries a methionine rather than a formylmethionine
The Translation Initiation Stage
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The start codon for eukaryotic translation is AUG
Ribosome scans from the 5 end of mRNA until it finds
the AUG start codon (not all AUGs can act as a start)
The consensus sequence for optimal start codonrecognition is shown here
Start codon
G C C (A/G) C C A U G G-6 -5 -4 -3 -2 -1 +1 +2 +3 +4
Most important positions for codon selection
These rules are called Kozaks rules
After Marilyn Kozak who first proposed them
With that in mind, the start codon for eukaryotic
translation is usually the first AUG after the 5 Cap!
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Translational initiation in eukaryotes can be
summarized as such:
An initiation factor protein complex (eIF4) binds to the 5
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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During this stage, amino acids are added to the
polypeptide chain, one at a time
This process, though complex, can occur at a
remarkable rate In bacteria 15-20 amino acids per second
In eukaryotes 2-6 amino acids per second
The Translation Elongation Stage
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Figure 13.19
The 23S rRNA (a
component of the large
subunit) is the actual
peptidyl transferase
Thus, the ribosome
is a ribozyme!
3
P site
Codon 3Codon 4
mRNA
E siteA site
aa1aa2
aa3 Ribosome
aa1aa2aa3
EAP
aa4
A charged tRNA bindsto the A site. EF-Tufacilitates tRNA bindingand hydrolyzes GTP.
Peptidyltransferase, which
is a component of the 50Ssubunit, catalyzes peptidebond formation between thepolypeptide and the aminoacid in the A site. Thepolypeptide is transferred
to the A site.
5
5
3
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Figure 13.19
tRNAs at the P and
A sites move into
the E and P sites,
respectively
Codon 4Codon 5
Codon 335
aa1aa2aa3aa4
aa1aa2
aa3
E A
A
Codon 4
Codon 5Codon 33
5
aa1aa2aa3
aa4
E
A
P
P
aa4
This process is repeated, again and
again, until a stop codon is reached.
An unchargedtRNA is releasedfrom the E site.
The ribosome translocates1 codon to the right. This
translocation is promotedby EF-G, which hydrolyzesGTP.
53
E P
40
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16S rRNA (a part of the 30S ribosomal subunit) plays
a key role in codon-anticodon recognition
If incorrect tRNA bound at the A site
It will prevent elongation until the mispaired tRNA is released
This phenomenon is termed the decoding function
of the ribosome
It is important in maintaining the high fidelity of mRNAtranslation
Error rate: 1 mistake per 10,000 amino acids added
The Translation Elongation Stage
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42
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The final stage occurs when a stop codon is
reached in the mRNA
In most species there are three stop ornonsense codons
UAG UAA
UGA
These codons are not recognized by tRNAs, but byproteins called release factors
The 3-D structure of release factors mimics that of
tRNAs
The Translation Termination Stage
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Bacteria have three release factors
RF1, which recognizes UAA and UAG
RF2, which recognizes UAA and UGA RF3, which does not recognize any of the three codons
It binds GTP and helps facilitate the termination process
Eukaryotes only have one release factor eRF, which recognizes all three stop codons
The Translation Termination Stage
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Figure 13.20
35
Stop codonin A site
tRNA in Psite carriescompletedpolypeptide
E A
35
E A
mRNA A release factor (RF) binds to the A site.
The polypeptide is cleaved from the tRNAin the P site. The tRNA is then released.
The ribosomal subunits, mRNA, andrelease factor dissociate.
Releasefactor
3
3
5
5
50S subunit 30S subunit
mRNA
P
P
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46
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Bacteria lack a nucleus
Therefore, both transcription and translation occur in the
cytoplasm
As soon an mRNA strand is long enough, a ribosome will
attach to its 5 end
So translation begins before transcription ends
This phenomenon is termed coupling
A polyribosome orpolysome is an mRNA transcript that has
many bound ribosomes in the act of translation
Bacterial Translation Can Begin
Before Transcription Is Completed
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Coupling between transcription and translation in bacteria