Transcript Brooker Chapter 13

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Genetics: Analysis and Principles
Robert J. Brooker
CHAPTER 13
TRANSLATION OF mRNA
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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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13.1 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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Archibald Garrod

First to propose (at the beginning of the 20th century)
a relationship between genes and protein production

Garrod studied patients who had defects in their
ability to metabolize certain compounds

He was particularly interested in alkaptonuria


Patients bodies accumulate abnormal levels of
homogentisic acid (alkapton)
Disease characterized by

Black urine and bluish black discoloration of cartilage and skin
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Archibald Garrod

He proposed that alkaptonuria was due to a missing
enzyme, namely homogentisic acid oxidase

Garrod also knew that alkaptonuria follows a
recessive pattern of inheritance

He proposed that a relationship exists between the
inheritance of the trait and the inheritance of a
defective enzyme

He described the disease as an inborn error of metabolism
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Figure 13.1
Metabolic pathway of phenylalanine metabolism and related
genetic diseases
13-6
Beadle and Tatum’s Experiments

In the early 1940s, George Beadle and Edward
Tatum were also interested in the relationship
among genes, enzymes and traits

They specifically asked this question


Is it One gene–one enzyme or one gene–many enzymes?
Their genetic model was Neurospora crassa (a
common bread mold)

Their studies involved the analysis of simple nutritional
requirements
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Beadle and Tatum’s Experiments

They analyzed more than 2,000 strains that had
been irradiated to produce mutations

They found three strains that were unable to grow
on minimal medium (Table 13.1)

However, in each case, growth was restored if only a
single vitamin is added to the minimal medium



1st strain  Pyridoxine
2nd strain  Thiamine
3rd strain  p-aminobenzoic acid
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Beadle and Tatum’s Experiments

In the normal strains, these vitamins were
synthesized by cellular enzymes


In the mutant strains, a genetic defect in one gene
prevented the synthesis of one protein required to
produce that vitamin
Beadle and Tatum’s conclusion: A single gene
controlled the synthesis of a single enzyme

This was referred to as the one gene–one enzyme theory
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13-10
Beadle and Tatum’s Experiments

In later decades, this theory had to be modified in
two ways

1. Enzymes are only one category of proteins

2. Some proteins are composed of two or more different
polypeptides


The term polypeptide denotes structure
The term protein denotes function
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The Genetic Code

Translation involves an interpretation of one
language into another


This relies on the genetic code


In genetics, the nucleotide language of mRNA is
translated into the amino acid language of proteins
Refer to Table 13.2
The genetic information is coded within mRNA in
groups of three nucleotides known as codons
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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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
Figure 13.2 provides an overview of gene expression
Figure 13.2
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Evidence that the Genetic Code is
Read in Triplets

The first such evidence came in 1961 from studies of Francis
Crick and his colleagues

These studies involved the isolation of phage T4 mutants




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
 rII phages were recovered and analyzed
These mutants were then re-exposed to proflavin
+
 r phages were recovered and analyzed
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Evidence that the Genetic Code is
Read in Triplets

The strains were analyzed using recombinational methods


These were described in Chapter 6
As shown in the hypothetical example of Table 13.4, the
wild-type plaque morphology is restored by

1. A (+) and a (-) mutation that are close to each other


2. Three (-)(-)(-) mutation combinations


AND MORE IMPORTANTLY
NOT two!
These results are consistent with the idea that the genetic
code is read in multiples of three nucleotides
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13-18
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13-19
Experiment 13A: Synthetic RNA
Helped Decipher the Genetic Code

The genetic code was deciphered in the early 1960s


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
 However, they made a major advance


They discovered that addition of synthetic RNA to DNase-treated
extracts restores polypeptide synthesis
Moreover, they added radiolabeled amino acids to these
extracts

Thus, the polypeptides would be radiolabeled and easy to detect
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13-20

To make synthetic RNA, the enzyme polynucleotide
phosphorylase was used


In the presence of excess ribonucleoside diphosphates (NDPs), it
catalyzes the covalent linkage of ribonucleotides into RNA
 Since it does not use a template, the order of nucleotides is random
An experimenter can control the amounts of nucleotides added

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%
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The Hypothesis

The sequence of bases in RNA determines the
incorporation of specific amino acids in the
polypeptide

The experiment aims to help decipher the relationship
between base composition and particular amino acids
Testing the Hypothesis

Refer to Figure 13.3
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13-22
Figure 13.3
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The Data
Radiolabeled Amino
Acid Added
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
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Interpreting the Data
Due to two codons:
GGG (34%) and GGU (15%)
Radiolabeled Amino
Acid Added

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
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 this is but one example of one type of experiment
that helped decipher the genetic code
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13-25
RNA Copolymers Helped to Crack
the Genetic Code

In the 1960s, Gobind Khorana and his collaborators
developed a novel method to synthesize RNA



They first created short RNAs (2 to 4 nucleotide 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 13.3

Refer to Table 13.5
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13-27
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 13.4
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13-28

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 13.4

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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
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 13.5
They are often buried
within the interior of a
folded protein
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13-30

Nonpolar and charged amino acids are hydrophilic

They are more likely to be on the surface of the protein
Figure 13.5
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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

These are stabilized by the formation of hydrogen bonds

Refer to Figure 13.6b

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13-32
Levels of Structures in Proteins

The short regions of secondary structure in a protein
fold into a three-dimensional tertiary structure



Refer to Figure 13.6c
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
Refer to Figure 13.6d
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13-33
A protein
subunit
Figure 13.6
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13-34
Functions of Proteins

To a great extent, the characteristics of a cell depend on the
types of proteins its makes

Proteins can perform a variety of functions
 Refer to Table 13.6

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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Figure 13.7
A comparison of phenotype and genotype at the molecular, organismal
and cellular levels
13-37
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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13-38
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
Proline
anticodon
Figure 13.8
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13-39
Experiment 13B: The Adaptor
Hypothesis Put to the Test

In 1962, François Chapeville and his colleagues conducted
studies to test the adaptor hypothesis

According to the hypothesis, the amino acid attached to tRNA
is not directly involved in codon recognition


Therefore, the alteration of an amino acid already attached to tRNA
should cause that altered amino acid to be incorporated into the
polypeptide instead of the normal amino acid
Example: Cysteine on a tRNAcys is changed to alanine
cys will add alanine instead of the usual
 Therefore, the tRNA
cysteine
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13-40

Chapeville had a chemical that converted cysteine to alanine


Raney nickel
The experiment made use of a cell-free translation system
similar to the one used by Nirenberg

Refer to Figure 13.3

Chapeville used an mRNA template that contained only U and G


Therefore, it could only contain the following eight codons
UUU = phenylalanine
GUU = valine
UUG = leucine
GUG = valine
UGU = cysteine
GGU = glycine
UGG =tryptophan
GGG = glycine
Note: One cysteine codon and no alanine codons
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13-41
The Hypothesis

Codon recognition is dictated only by the tRNA

The chemical structure of the amino acid attached to
tRNA does not play a role
Testing the Hypothesis

Refer to Figure 13.9
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13-42
Figure 13.9
13-43
Figure 13.9
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The Data
Relative Amount of Radiolabeled
Amino Acid Incorporated into
Polypeptide (cpm)*
Conditions
Control, untreated tRNA
Raney nickel-treated tRNA
Cysteine
Alanine
Total
2,835
83
2,918
990
2,020
3,010
*Cpm is counts per minute of radioactivity in the sample
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13-45
Interpreting the Data
Relative Amount of Radiolabeled
Amino Acid Incorporated into
Polypeptide (cpm)*
Conditions
Control, untreated tRNA
Raney nickel-treated tRNA
Cysteine
Alanine
Total
2,835
83
2,918
990
2,020
3,010
Expected result
since only
radiolabeled
cysteine was added
Probably a result
of cysteine
contamination
*Cpm is counts per minute of radioactivity in the sample
About a third of the
tRNAcys did not react
with the Raney nickel
Large amount of incorporated
alanine even though template
mRNA lacks alanine codons
Overall, these results support the adaptor hypothesis
tRNAs act as adaptors to carry the correct amino acid to
the ribosome based on their anticodon sequence
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13-46
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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13-47
Found in all tRNAs
Not found in all tRNAs
Other variable sites are
shown in blue as well







Figure 13.10 Structure of tRNA
The modified bases are:
I = inosine
mI = methylinosine
T = ribothymidine
UH2 = dihydrouridine
m2G = dimethylguanosine
y = pseudouridine
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