Transcript Brooker Chapter 13 - Volunteer State Community College

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Genetics: Analysis and Principles
Robert J. Brooker
CHAPTER 13 Part 1
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
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13-2
13.1 THE GENETIC BASIS FOR
PROTEIN SYNTHESIS

Genes that encode polypeptides are termed
structural genes

These are transcribed into messenger RNA (mRNA)

The translation of the mRNA codons into amino
acid sequences leads to the synthesis of proteins

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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13-3
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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13-5
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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13-7
Beadle and Tatum’s evidence for the one gene-one enzyme hypothesis
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
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13-9
Beadle and Tatum’s Experiments

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

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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13-10
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
The genetic information is coded within mRNA in
groups of three nucleotides known as codons
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13-12
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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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13-15

Figure 13.2 provides an overview of gene expression
Figure 13.2
13-16
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
 Addition of synthetic RNA to DNase-treated extracts
generates 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
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13-21

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%
13-21
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
13-23
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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13-24
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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13-26
Deciphering the code
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13-27
Nirenberg & Leder: Triplet Binding Assay
Triplet Binding Assay
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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13-29

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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13-31
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

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13-32
Protein Structure: Tertiary structure
CH
CH2
Hydrogen
bond
H3C
CH3
H3C
CH3
CH
O
H
O
Hydrophobic
interactions and
van der Waals
interactions
OH C
CH2
CH2 S S CH2
Disulfide bridge
O
CH2 NH3+ -O C CH2
Ionic bond
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Polypeptide
backbone
Levels of Structures in Proteins

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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13-33
A protein
subunit
Figure 13.6
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13-36
Figure 13.7
A comparison of phenotype and genotype at the molecular, organismal
and cellular levels
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