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INTRODUCTION
• 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
– 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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THE GENETIC BASIS FOR PROTEIN SYNTHESIS
• Proteins are the active participants in cell
structure and function
• Genes that encode polypeptides are termed
structural genes
– These are transcribed into messenger RNA (mRNA)
• The main function of the genetic material is to
encode the production of cellular proteins
– In the correct cell, at the proper time, and in suitable
amounts
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The Genetic Code

Translation involves an interpretation of one
language into another

In genetics, the nucleotide language of mRNA is
translated into the amino acid language of proteins

This relies on the genetic code

The genetic information is coded within mRNA in
groups of three nucleotides known as codons
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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

Special codons:

AUG (which specifies methionine) = start codon




UAA, UAG and UGA = termination, or stop, codons
The code is degenerate

More than one codon can specify the same amino acid


For example: GGU, GGC, GGA and GGG all code for lysine
In most instances, the third base is the degenerate base


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

Only a few rare exceptions have been noted
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
An overview of gene expression
Note that the start codon sets the
reading frame for all remaining codons
A Polypeptide Chain Has Directionality

Polypeptide synthesis has a directionality that parallels the 5’
to 3’ 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
 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

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R1 O
H3N+
C
C
H
R2 O
N
C
H
H
R1 O
H3N+ C
C
H
R3 O
C
C
H
H
+
N
C
C
H
H
O–
R2 O
N
R4 O
H3N+
R3 O
C
N
C
H
H
C
C
C
H
O–
R4 O
N
C
C + H2O
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
H3C
H
+
Amino- H3N
terminal
end
C
C
H
O
Methionine
N
H
C
C
H
O
Serine
SH
CH3
CH
CH2
H
N
C
C
H
O
Valine
N
CH2
H
C
C
H
O
Tyrosine
N
C
C
H
O
O– Carboxylterminal
end
Cysteine
Peptide bonds
5′
AUG
AGC
GU U
UAC
UGC
Sequence in mRNA
(b) Directionality in a polypeptide and mRNA
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3′
•
There are 20 amino acids that may be found in polypeptides
– Each contains a different side chain, or R group
– Each R group has its own particular chemical properties

Nonpolar amino acids are
hydrophobic

They are often buried
within the interior of a
folded protein
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
Nonpolar and charged amino acids are hydrophilic

They are more likely to be on the surface of the protein
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Levels of Structures in Proteins

There are four levels of structures in proteins





1.
2.
3.
4.
Primary
Secondary
Tertiary
Quaternary
A protein’s primary structure is its amino acid
sequence
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• Within the cell, the
protein will not be
found in this linear
state
– Rather, it will adapt
a compact 3-D
structure
– Indeed, this folding
can begin during
translation
129 amino
acids long
• The progression from
the primary to the 3-D
structure is dictated by
the amino acid
sequence within the
polypeptide
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Levels of Structures in Proteins


The primary structure of a protein folds to form
regular, repeating shapes known as secondary
structures
There are two types of secondary structures

a helix
b sheet

Certain amino acids are good candidates for each structure

These are stabilized by the formation of hydrogen bonds

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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
Proteins made up of two or more polypeptides have
a quaternary structure

This is formed when the various polypeptides associate
together to make a functional protein
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Functions of Proteins

To a great extent, the characteristics of a cell depend on the
types of proteins it makes

Proteins can perform a variety of functions

A key category of proteins are enzymes


Accelerate chemical reactions within a cell
Can be divided into two main categories
 Anabolic enzymes  Synthesize molecules and macromolecules
 Catabolic enzymes  Break down large molecules into small ones

Important in generating cellular energy
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STRUCTURE AND FUNCTION OF tRNA
• In the 1950s, Francis Crick and Mahon Hoagland
proposed the adaptor hypothesis
– tRNAs play a direct role in the recognition of codons in
the mRNA
• In particular, the hypothesis proposed that tRNA
has two functions
– 1. Recognizing a 3-base codon in mRNA
– 2. Carrying an amino acid that is specific for that codon
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Recognition Between tRNA and mRNA

During mRNA-tRNA recognition, the anticodon in
tRNA binds to a complementary codon in mRNA
tRNAs are named
according to the
amino acid they bear
The anticodon is
anti-parallel to
the codon
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tRNAs Share Common Structural Features

The secondary structure of tRNAs exhibits a
cloverleaf pattern

It contains


Three stem-loop structures; Variable region
An acceptor stem and 3’ 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

More than 80 of these can occur
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Found in all tRNAs
Not found in all tRNAs
Other variable sites are
shown in blue as well
•
•
•
•
•
•
•
Structure of tRNA
The modified bases are:
I = inosine
mI = methylinosine
T = ribothymidine
UH2 = dihydrouridine
m2G = dimethylguanosine
y = pseudouridine
Charging of tRNAs

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
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Charging of tRNAs

The aminoacyl-tRNA synthetases are responsible for the
“second genetic code”

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

Many modified bases are used as markers
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The amino acid is
attached to the 3’ end
by an ester bond
tRNAs and the Wobble Rule

The genetic code is degenerate


With the exception of serine, arginine and leucine, this
degeneracy always occurs at the codon’s third position
To explain this pattern of degeneracy, Francis Crick proposed
the wobble hypothesis
 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

Thus tolerating certain types of mismatches
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RIBOSOME STRUCTURE AND ASSEMBLY
• Translation occurs on the surface of a large
macromolecular complex termed the ribosome
• Bacterial cells have one type of ribosome
– Found in their cytoplasm
• Eukaryotic cells have two types of ribosomes
– One type is found in the cytoplasm
– The other is found in organelles
• Mitochondria ; Chloroplasts
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RIBOSOME STRUCTURE AND ASSEMBLY
• 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
– Each subunit is formed from the assembly of
• Proteins
• rRNA
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Functional Sites of Ribosomes

During bacterial translation, the mRNA lies on the
surface of the 30S subunit


As a polypeptide is being synthesized, it exits through a
hole within the 50S subunit
Ribosomes contain three discrete sites



Peptidyl site (P site)
Aminoacyl site (A site)
Exit site (E site)
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STAGES OF TRANSLATION
• Translation can be viewed as occurring in three
stages
– Initiation
– Elongation
– Termination
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Initiator tRNA
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The Translation Initiation Stage

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 to
N-formylmethionine
The start codon is AUG, but in some cases GUG or UUG

In all three cases, the first amino acid is N-formylmethionine
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
The binding of mRNA to the 30S subunit is facilitated by a
ribosomal-binding site or Shine-Dalgarno sequence

This is complementary to a sequence in the 16S rRNA
Component of the
30S subunit
Hydrogen bonding
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IF1 and IF3 bind to the 30S subunit.
IF3
30S subunit
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′
IF1
IF1
Start
Shinecodon
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.
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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.
70S initiation
complex
tRNAfMet
E
5′
P
A
70S
initiation
complex
3′
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This marks the
end of the first
stage
The Translation Initiation Stage

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
The initiator tRNA is designated tRNAmet

It carries a methionine rather than a formylmethionine
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
The start codon for eukaryotic translation is AUG

Scanning ribosome may pass over the first AUG

But in most cases, 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:
 A number of initiation factors bind 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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The Translation Elongation Stage

During this stage, the amino acids are added to the
polypeptide chain, one at a time

This process, though complex, can occur at a
remarkable rate


In bacteria  15-18 amino acids per second
In eukaryotes  6 amino acids per second
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The 23S rRNA (a component of
the large subunit) is the actual
peptidyl transferase
Thus, the ribosome
is a ribozyme!
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tRNAs at the P and A
sites move into the E
and P sites,
respectively
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The Translation Elongation Stage

16S rRNA (a part of the 30S ribosomal subunit) plays a key
role in codon-anticodon recognition

It can detect an 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 in mRNA
translation

Error rate: 1 mistake per 10,000 amino acids added
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The Translation Termination Stage

The final stage occurs when a stop codon is
reached in the mRNA

In most species there are three stop or nonsense codons




UAG
UAA
UGA
These codons are not recognized by tRNAs, but by
proteins called release factors

Indeed, the 3-D structure of release factors mimics that of tRNAs
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The Translation Termination Stage

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
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Bacterial Translation Can Begin Before
Transcription Is Completed

Bacteria lack a nucleus


As soon an mRNA strand is long enough, a ribosome will
attach to its 5’ end



Therefore, both transcription and translation occur in the cytoplasm
So translation begins before transcription ends
This phenomenon is termed coupling
A polyribosome or polysome is an mRNA transcript that has
many bound ribosomes in the act of translation
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Coupling between transcription and translation in bacteria
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tRNAs that can recognize the same
codon are termed isoacceptor tRNAs

inosine
5-methyl-2-thiouridine
5-methyl-2’-O-methyluridine

2’-O-methyluridine

5-methyluridine



5-hydroxyuridine

lysidine
Wobble position and base pairing rules
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Recognized
very poorly by
the tRNA