Download In experiments with a 3 base codon system it was shown that the

In experiments with a 3 base codon system it
was shown that the code was a nonoverlapping code, meaning that the bases in
one codon were not part of another codon.
The presence of 64 codons for 20 amino acids
allows for some redundancy, meaning that an
amino acid may have more than one codon
coding for that amino acid.
The fact that the codons for an amino acid
were similar lead to the proposal by Crick of
the ‘wobble’ hypothesis. In this hypothesis
the specificity of the code is more in the first
two bases allowing for variation in pairing at
the third base without changing the amino
acid .
51
example:
proline - CCU
CCC
CCA
CCG
This system could increase the speed of
protein synthesis and reduce errors.
Summary - Genetic Code
1) 3 bases per codon
2) non-overlapping code
3) some degeneracy or redundancy in the
code
4) can have ‘wobble’ for amino acid
specificity with a greater specificity for the
first two bases of a codon
5) code is almost universal, a major reason
for genetic engineering
52
Conversion of the genetic information into a
product requires the translation of the
nucleotide sequence in DNA to an amino acid
sequence in a protein.
This concept of one gene  one enzyme was
first proposed by Beadle and Tatum (1941)
Problem with a direct translation (at least in
eukaryotes) DNA is found in the nucleus
while protein synthesis occurs in the
cytoplasm.
An intermediary is needed - Ribonucleic Acid
RNA
RNA is found in both the nucleus and
cytoplasm and is similar to DNA since it also
has four bases: A, U, G, C.
53
Translation - The formation of a polypeptide
with the amino acid sequence directed by the
nucleotide sequence of a specific RNA
molecule (mRNA)
There are 3 types of RNA needed for
translation:
 messenger RNA (mRNA)
 transfer RNA (tRNA)
 ribosomal RNA (rRNA)
54
Messenger RNA
 large molecular weight (500,000 +)
 intermediate carrier of the genetic code
 relatively short-lived but will vary among
genes and between prokaryotes and
eukaryotes
 may be translated many times
 2 to 10% of cellular RNA
 amount of modification required prior to
translation differs between prokaryotes and
eukaryotes
55
Difference in mRNA between eukaryotes and
prokaryotes is the processing required after
transcription.
Eukaryotes
Prokaryotes
DNA
DNA


transcription
hnRNA
mRNA

processed
in nucleus
ready for
translation
mRNA

add
cap


excised add
introns tail
ready for translation
Eukaryotic gene
56
exon
intron
exon
intron
exon
exon - region that codes for part of the gene
intron- region that does not code for part of
the gene
How the presence of introns was detected:
expected
observed
mRNA
ssDNA
mRNA
ssDNA
Why the need for processing?
57
1) Remove the RNA that does not code for the
specific polypeptide.
Lead to the idea of split genes - where there
are regions within the DNA sequence of a
gene that does not appear in the final
product.
2) Place a methylated guanine (mG) on the 5’
end of the mRNA.
No phosphate
mG
cap is
attached in
reverse
cap is needed for attachment of the small
ribosomal sub-unit during translation.
58
3) Polyadenine tail is added after
transcription by a specific adenine
polymerase.
The poly-A tail may vary in length and
may act as an attachment site for proteins
that protect or assist in the transport of the
mRNA through the cytoplasm.
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Transfer RNA (tRNA)
 low molecular weight (25,000 +)
 70 to 90 nucleotides in length
 contains modified bases:
examples - inosinic acid
hypothxanthine
ribothymidylic acid
pseudouridine
 modification of the bases occurs posttranscription.
 some tRNA require processing.
 makes up 10-15% of cellular RNA
 each tRNA is specific for one amino acid
 there may be more than one tRNA for each
amino acid
60
‘Wobble’ in the anticodon loop of tRNA
nucleotide at
5’ end of the
anticodon
nucleotide at
3’ end of codon
G
can pair with
U or C
C
can pair with
G
A
can pair with
U
U
can pair with
A or G
I (inosine) can pair with A, U or C
The wobble makes it possible to have only 32
tRNA’s for the 61 possible codons.
61
Ribosomal RNA (rRNA)
 variable molecular weights
 variable number of nucleotides (120 to
4800)
 70 to 80 percent of the cellular RNA
 Differs between prokaryotes and
eukaryotes in size
Prokaryote
Eukaryote
70S monosome
80S monosome


50S large 30S small
60S large 40S small




23S rRNA 16S rRNA
28S rRNA 18S rRNA
5S rRNA
5.8S rRNA
5.0S rRNA
31 proteins 21 proteins 50 proteins 33 proteins
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Translation requires:
 61 types of tRNA
 20 types of aminoacyl tRNA synthetases
 Large ribosomal subunit
 Small ribosomal subunit
 m RNA
 modified met - tRNA
 Initiation, Elongation, and Termination
factors
63
Translation
 Prior to translation
Activation of tRNA
amino acid + ATP
 aminoacyl tRNA
synthetase
amino acid - AMP + specific tRNA
 aminoacyl tRNA
synthetase
amino acid - tRNA + AMP
64
Characteristics of Translation
1. mRNA is translated 5’  3’
2. Protein synthesis occurs
N-terminal  C-terminal
3. Leader section of mRNA is not translated
4. Rate of translation 4-15 amino acids/sec
5. Many ribosomes can translate same mRNA
(polysome or polysome complex)
6. Modified met-tRNA for first amino acid
prokaryotes  N-formyl methionine
eukaryotes  ‘initiator’ methionine
65
Initiation of Translation
 small ribosomal subunit
 large ribosomal subunit
 Mg++
 GTP
 mRNA
 initiation factors IF1, IF2, and IF3
 fmet-tRNA
66
Steps in the initiation of translation:
1) Small subunit attaches to 5’ end of mRNA
at the Shine-Dalgarno sequence in the
leader sequence assisted by IF3.
2) fmet-tRNA attaches to peptidyl site of the
small ribosomal subunit assisted by IF2.
3) Large subunit attaches to the small subunit
67
Elongation of the polypeptide chain
1) Next amino acid - tRNA enters the
aminoacyl site assisted by elongation factor
EF-Tu
2) Peptide bond formation occurs, catalyzed
by peptidyl transferase
3) Release of fmet-tRNA
4) Translocation of the ribosome to the next
codon assisted by elongation factor
EF-G opening up the aminoacyl site
5) Next amino acid - tRNA enters the
aminoacyl site assisted by elongation factor
EF-Tu
68
Termination of Translation
1) The ribosome reaches one of three
termination codons (UAG, UAA, UGA)
2) Release factor RF1 or RF2 binds to the
open aminoacyl site
3) Disassociation occurs resulting in the
separation of the following components:
mRNA
polypeptide
small ribosomal subunit
large ribosomal subunit
final tRNA
69
Translation in Eukaryotes
Very similar to prokaryotic translation
differences:
 5’ cap is the attachment site for the small
subunit of the ribosome.
 Formyl methionine is not required to start
translation. Instead a unique initiator
tRNAmet is required. The start codon is still
AUG.
 A greater number of initiation, elongation
and termination factors are required.
70
Products of Translation
polypeptide = protein
Classes of proteins
 enzymes
 receptor proteins
 transport proteins
 structural proteins
 nucleic acid binding proteins
 ribosomal proteins
 storage proteins
71
Protein Structure
Structure and function of a protein is
controlled by the sequence of the amino acids
and the interaction of the amino acids within
the polypeptide and with amino acids in other
polypeptides.
Primary structure: linear sequence of amino
acids
Secondary structure: interaction of amino
acids in the polypeptide in the form of
hydrogen bonds that result in the folding of
the polypeptide into various
shapes/structures.
Examples:
 helix
 pleated sheets
72
Tertiary structure: additional folding of the
polypeptide by covalent bonds forming
between amino acid side groups.
The folding due to covalent bonds will be
more permanent than those found in the
secondary structure, why?
cyst.
S
S
cyst.
Quaternary structure: interaction between
polypeptides.
Example:
enzymes with multiple sub-units
73
Structure of the protein dictates its function.
Change the amino acid sequence and you
may change the structure of the protein.
A change in structure can lead to reduced
functionality or non-functionality.
So changes in the base sequence of the DNA
within a gene can change the functionality of
the gene product if the change results in an
amino acid(s) change in a critical location of
the polypeptide.
74
Gene Mutations
Gene mutations are changes in the DNA
sequence that cause a detectable change in a
gene (i.e. change the expression of a gene).
Gene mutations can occur spontaneously or
can be induced.
There are two basic types of gene mutations:
 base deletions or additions
 base change
Base deletions or additions cause a frameshift
mutation because they change the reading
frame of the gene.
Add or delete 1 - 2 bases  a nonsense
mutation
Add or delete 3 bases  a missense mutation
Base changes will cause a missense mutation
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A single base change can have no impact or a
major impact depending on:
1) If the codon changed effects a critical
amino acid in the polypeptide
2) if the codon is changed to a stop codon
3) if the codon that is changed is the start
codon
Base change mutations can either be a
forward mutation or a reversion mutation.
The forward mutation changes the functional
form of the gene to a mutant form.
A reversion mutation restores either the
original DNA sequence of a gene or restores
the function of the gene. Example of this is if
the forward mutation was a base deletion
(frameshift) a base insertion would be a
reversion mutation (restores normal reading
frame)
76
Gene mutations can either be spontaneous or
are induced.
Spontaneous mutations are ones that occur
naturally due to an error in replication.
A replication error can occur due to a
mutation in the proof-reading ability of DNA
pol I or III or due to a change in base
configuration.
A change in the base configuration is called a
tautomeric shift where a base goes from the
keto to the enol form.
For example, if a tautomeric shift occurs in a
thymine, it will be paired to guanine instead
of adenine. If adenine undergoes a
tautomeric shift it will now be read as a
guanine and be paired with cytosine instead
of thymine.
77
Induced mutations are the result of an
artificial factor.
Gene mutations can be induced by:
 sunlight (u.v. light)
 radiation
 chemicals
Sunlight - u.v. light
U.v. light induces mutations by causing
adjacent thymines in a DNA strand to
connect forming a thymine dimer.
--------------------T T ------------------------------------------A A ----------------------At replication the presence of a thymine
dimer can cause errors in reading by the
DNA polymerase leading to the wrong base(s)
being inserted.
78
There are two repair systems for u.v. light
induced mutations:
 light activated repair
 dark repair
The light activated repair requires energy
from light to work and is called
photoreactivation repair catalyzed by the
enzyme photolyase.
Dark repair is an excision/repair system
where an endonuclease first removes the
dimer, DNA pol I replaces the missing bases ,
and ligase makes the final phosphodiester
bond.
What happens without a repair system?
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Humans - xeroderma pigmentosum
 skin cancer
Radiation
Radiation can cause damage in two ways.
A high energy particle hits a DNA strand
removing a base, bases, or whole sections of
the DNA causing a break in the DNA.
A second way to cause damage is the
production of free radicals (molecules lacking
an electron) that can react with DNA causing
a break in the DNA strand or modifying
bases within the strand.
The rate of mutation increases with the level
of the dose.
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Chemical mutagens
Chemical mutagens will cause a change in a
base changing how it will pair in the next
cycle of replication. This will cause a change
in a codon, i.e. ATT  GTT
There are two types of base changes,
transitions and transversions.
Transition:
 purine  purine, ex. AG
 pyrimidine  pyrimidine, ex. CT
Transversion:
 purine  pyrimidine, ex. AC
- pyrimidine  purine, ex. TG
Examples of chemical mutagens
81
1. base analogs - where a chemical is similar
to a base but pairs differently
example 1 - 5-bromouracil (BU) which is an
analog to thymine.
If you were to go through a series of
replications the base change would occur in
this way.
A
A
G
G



T
BU
BU
C
So the change is a transition, thymine to
cytosine
example 2 - aminopurine (AP)
an analog to adenine
T
T

A
C

AP
C

AP
G
82
so the change is a transition, adenine to
guanine
2. Deamination - where a chemical will
modify the base structure changing pairing
in the next cycle of replication.
Example - nitrous acid - removes amino
groups
adenine is changed to hypoxanthine (H)
which pairs to cytosine instead of thymine.
A
H

T

C
amino group
G

C
cytosine is changed to uracil which pairs with
adenine instead of guanine.
83
C
U

G

A
amino group
T

A
3. alkylating agents - where a chemical will
change the base structure by adding a
methyl group, changing base pairing.
Example - ethyl methanesulfonate (EMS) will add a methyl group to guanine making it
a base analog to thymine.
G
m
G

C

A
methyl group
T

A
84
Testing for chemical mutagens
As new chemicals are developed it is
important to determine if they pose a risk to
humans/the environment as a mutagen or a
carcinogen.
What is the difference between a mutagen
and a carcinogen?
What is the challenge in testing if a chemical
is a mutagen?
The Ames test was developed to determine if
a chemical could induce either frameshift or
base change mutations.
The test used bacteria that contained either a
frameshift mutation or a base change
mutation making it dependent on a nutrient
supplement in the media for survival (called
an auxotroph).
85
If bacteria are detected after treatment with
the chemical that do not require the nutrient
supplement for survival (become prototroph
or wild-type strain) at a higher rate then
normal (due to natural errors in replication)
the chemical is considered a mutagen.
Why is it easier to detect mutations from
auxotrophs to prototrophs then prototrophs
to auxotrophs?
What has been the complaint about testing
chemicals in this way?
86