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Genetics: Analysis and Principles
Robert J. Brooker
CHAPTER 13
TRANSLATION OF mRNA
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13-13

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


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

Refer to Table 13.3
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13-14
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
Figure 13.2 provides an overview of gene expression
Figure 13.2
Note that the start codon sets the
reading frame for all remaining
codons
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Carboxyl group
Amino group
Condensation
reaction releasing a
water molecule
Figure 13.5
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13-30
N terminal
Figure 13.5
C terminal
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
There are 20 amino acids that may be found in polypeptides
 Each contains a different side chain, or R group

Nonpolar amino acids are
hydrophobic

Figure 13.6
They are often buried
within the interior of a
folded protein
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13-32

Nonpolar and charged amino acids are hydrophilic

They are more likely to be on the surface of the protein
Figure 13.6
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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

Refer to Figure 13.7
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13-34

The amino acid
sequence of the
enzyme
lysozyme
Within the cell, the
protein will not be
found in this linear
state
 Rather, it will adapt
a compact 3-D
structure

129 amino acids
long
Figure 13.7

Indeed, this folding
can begin during
translation
The progression from
the primary to the 3-D
structure is dictated by
the amino acid
sequence within the
polypeptide
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13-35
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

Refer to Figure 13.8

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13-36
Levels of Structures in Proteins

The short regions of secondary structure in a protein
fold into a three-dimensional tertiary structure




Refer to Figure 13.8
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


This is formed when the various polypeptides associate
together to make a functional protein
Refer to Figure 13.8
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13-37
Figure 13.8
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13-38
13.2 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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13-42
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
Figure 13.10
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13-43
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 60 of these can occur
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13-51
Found in all tRNAs
Not found in all tRNAs
Other variable sites are
shown in blue as well







Figure 13.12 Structure of tRNA
The modified bases are:
I = inosine
mI = methylinosine
T = ribothymidine
UH2 = dihydrouridine
m2G = dimethylguanosine
y = pseudouridine
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1.The acceptor stem is a 7-base pair (bp)
stem made by the base pairing of the 5'terminal nucleotide with the 3'-terminal
nucleotide (which contains the CCA 3'terminal group used to attach the amino
acid).
The CCA tail This sequence is important
for the recognition of tRNA by enzymes and
critical in translation. In prokaryotes,
In most prokaryotic tRNAs and eukaryotic
tRNAs, the CCA sequence is added during
processing and therefore does not appear
in the tRNA gene
The D arm is a 4 bp stem ending in a loop
that often contains dihydrouridine.
The anticodon arm is a 5-bp stem whose
loop contains the anticodon.
The T arm is a 5 bp stem containing the
sequence TΨC where Ψ is a
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

Refer to Figure 13.13
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13-53
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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13-54
The amino acid is
attached to the 3’ end
by an ester bond
Figure 13.13
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tRNAs and the Wobble Rule

As mentioned earlier, 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 in 1966 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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13-56
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
Figure 13.14 Wobble position and base pairing rules
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Recognized
very poorly by
the tRNA
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13.3 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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13-58
13.3 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
Figure 13.15 presents the composition of bacterial
and eukaryotic ribosomes
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13-59
Synthesis and assembly of all ribosome
components occurs in the cytoplasm
Note: S or Svedberg units
are not additive
Figure 13.15
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13-60
Synthesized in
the nucleus
Produced in the
cytosol
Formed in the
cytoplasm during
translation
The 40S and 60S subunits are
assembled in the nucleolus
Then exported to the cytoplasm
Figure 13.15
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13-61
Functional Sites of Ribosomes

During bacterial translation, the mRNA lies on the
surface of the 30S subunit


Ribosomes contain three discrete sites




As a polypeptide is being synthesized, it exits through a
hole within the 50S subunit
Peptidyl site (P site)
Aminoacyl site (A site)
Exit site (E site)
Ribosomal structure is shown in Figure 13.16
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13-62
Figure 13.16
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13-63
13.4 STAGES OF
TRANSLATION

Translation can be viewed as occurring in three
stages




Initiation
Elongation
Termination
Refer to 13.17 for an overview of translation
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13-64
Initiator tRNA
Figure 13.17
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13-65
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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13-66

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
Figure 13.19

Figure 13.18 outlines the steps that occur during
translational initiation in bacteria
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13-67
Figure 13.18
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13-68
The only charged
tRNA that enters
through the P site
70S initiation
complex
All others enter
through the A site
This marks the
end of the first
stage
Figure 13.18
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13-69
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
Refer to Table 13.7
The initiator tRNA is designated tRNAmet

It carries a methionine rather than a formylmethionine
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13-70

The start codon for eukaryotic translation is AUG


It is usually the first AUG after the 5’ Cap
The consensus sequence for optimal start codon
recognition is show here
Most important positions for codon selection


C C A U G G
-2 -1 +1 +2 +3 +4
These rules are called Kozak’s rules


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 5’ Cap!
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13-71

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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13-72
The Translation Elongation Stage

During this stage, the amino acids are added to the
polypeptide chain, one at a time


In bacteria  15-18 amino acids per second
In eukaryotes  6 amino acids per second
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13-73
The 23S rRNA (a component of
the large subunit) is the actual
peptidyl transferase
Thus, the ribosome
is a ribozyme!
Figure 13.20
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13-74
tRNAs at the P and A
sites move into the E
and P sites,
respectively
Figure 13.20
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13-75
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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13-76
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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13-77
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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Figure 13.21
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