Suppressor Mutations

Suppressor mutations are mutations that reverse or

cancel the effect of a pre-existing mutations resulting in
the wild-type or near wild-type phenotype.
There are two types of Suppressor Mutations:
Phenotypic Suppression: Non-Genetic changes that alter an
organisms phenotype resulting in a wild-type phenotype.
Genotypic Suppression: Reversal of a mutant phenotype by
through the action of a second mutation.

Review of Protein Synthesis

Watch this video and pay attention to these facts:
Translations reads triplet code (codons) from the mRNA.
tRNA have an anti-codon which is complimentary to the
codons on mRNA determining which amino acid is
incorporated into the growing peptide.
The resulting protein folds in a specific manner
depending on its amino acid sequence. Ultimately, it is
this folding, or mis-folding, that causes phenotypic
variations.

Review of Protein Synthesis

Phenotypic Suppression
Non-Genetic changes that alter an organisms phenotype
resulting in a wild-type phenotype.
Example: At low concentrations, the antibiotic
streptomycin can cause misreading of the mRNA by
distorting codon-anticodon interaction.
Misreading the mRNA can cause a different amino acid to
be added to the growing peptide rather than the amino
acid called for by the nucleotide sequence.

Phenotypic Suppression
This alternate amino acid sequence is reflected in the
protein folding.
This different folding of the protein can restore the
function of the protein to the wild type and yield the wildtype phenotype.
No change in the genetic code has occurred. The only
change is the amino acid sequence of the protein caused
by mis-reading the mRNA.

Genotypic Suppression
Reversal of the effects of a mutation through the action of
a second mutation.
Inheritable changes in the genetic code have occurred.
There are two basic types of genotypic suppression:
Intragenic suppression - compensatory mutation within the
same gene yielding the wild-type phenotype.
Extragenic suppression - compensatory mutation in another
(different) gene yielding the wild-type.

Intragenic Suppression
There are three types of intragenic suppression:
Intracodon suppression (Reversion) single base
substitution in the specific codon previously causing the mutant
phenotype.
Frameshift suppression nucleotide addition or deletion
restores the reading frame producing a protein that is close to
the wild-type yielding the wild-type phenotype.
Distant compensatory suppression a change in the amino
acid sequence of a protein occurs at some other location of the
protein but this new sequence allows folding of the protein in a
manner that restores the wild-type phenotype.

Intracodon Suppression

In the wild type, youll see that Arginine is the amino acid
that gives proper folding of the protein.
After mutation, Serine is placed in the protein where
arginine previously was causing protein misfolding and a
mutant phenotype.

Intracodon Suppression

A further base substitution occurs within (Intra) the codon

giving the wild-type phenotype.
In the first example, Arginine is placed back in the amino
acid sequence. The second example will substitute
Asparagine which is similar in structure to Arginine.
In either case, the protein will fold properly and give the
wild-type phenotype.

Frameshift Suppression
Reading Frame Suppression involves nucleotide addition
or deletion and restores the reading frame producing a
protein that is close to the wild-type yielding the wild-type
phenotype.
Below, a base addition has caused a shift in the reading
frame of the mRNA resulting in a mutant phenotype.
After the CGA, all amino acids added to the elongating
peptide will be incorrect and result in a mis-folded protein.

Frameshift Suppression

Deletion of a base restores the reading frame of the

mRNA and the amino acid sequence is mostly restored.
In this example, 2 of the amino acids will differ from the
wild-type sequence.
As long as they are not dramatically different in properties
relative to the original amino acids, the resulting protein
will fold correctly and yield the wild-type phenotype.

Distant Compensatory Suppression

Distant compensatory suppression a change in the
amino acid sequence of a protein occurs at some other
location of the protein but this new sequence allows
folding of the protein in a manner that restores the wildtype phenotype.
Mutant phenotypes are caused by mis-folded proteins
that are the result of changes in the genetic code.

Distant Compensatory Suppression

Extragenic Suppression
Extragenic (also known as Intergenic) suppression
relieves the effects of a mutation in one gene by a
mutation somewhere else within the genome. The second
mutation is not on the same gene as the original
mutation.
There are 2 basic types of extragenic suppressors:
Codon-specific suppressors
Polarity suppressors

Extragenic Suppression
Codon-specific suppressors (also called informational
suppressors) are mutant tRNAs that cause the occasional
insertion of a new amino acid at the site of the mutant
codon.
These mutations usually have a base substitution in the
Anticodon of the tRNA so it incorporates its amino acid at
the site of the mutant codon which was causing the
mutation.

Codon Specific Suppressors

There are three types of codon-specific suppressors:
Nonsense suppressors
Missense suppressors
Frameshift suppressors

Nonsense Suppressors
The most common Extragenic Suppression is the
suppression of a Nonsense codon.
Nonsense codons are triplet code sequences on a mRNA
that signals the termination of translation.
They are codons for which no normal tRNA molecule
exists. The presence of a nonsense codon causes
termination of translation yielding truncated proteins.
That is to say, there is no tRNA that has an anticodon that
is complimentary to the nonsense codon.

Nonsense Suppressors
Truncated proteins are missing all amino acids that would
normally be translated downstream of the nonsense
codon.
Truncated proteins will fold in an incorrect manner and
whatever function the wild type protein had will be lost.
There are three nonsense codons and they are called
amber(UAG) ochre(UAA) and opal (UGA).

Nonsense Suppressors

Nonsense Suppressors
Extragenic Suppressors, by definition, involve a gene
other than the gene carrying the original mutation.
Here, that gene will be one that normally encodes the
tryptophan tRNA molecule.
A suppressor mutation will occur in that gene such that a
base substitution takes place at the base which would
code the anticodon of the tRNA.
The anticodon is now different for that tRNA.

Nonsense Suppressors

Nonsense Suppressors
Previously, the protein was truncated due to the
premature nonsense (stop) codon.
Here, the nonsense codon was the amber (UAG) codon.
After mutation of the gene encoding the tRNA, the amber
codon can be read as a sense codon.
The protein can reach full length because an amino acid
(tryptophan) is now incorporated into the growing peptide
at a site that previously was a nonsense codon.

Nonsense Suppressors

Nonsense Suppressors
The protein can now be a full length translation and, as
long as the amino acid substituted into the peptide is not
dramatically different, the peptide will fold properly and
restore wild type function.
A secondary mutation in an unrelated tRNA gene results
in a suppressor tRNA charged with trp that can decode
amber (UAG) stop codons.

Missense Suppressors
Missense mutations involve base substitutions in a gene
encoding a mRNA where one codon is changed calling
for a wildly dissimilar amino acid at a specific place in the
peptide.
The protein loses function due to improper folding.
A new, mutant tRNA can be created to read that
missense codon and incorporate an amino acid into the
peptide that is similar to the wild type and allow
restoration of function through proper protein folding.

Frameshift Suppressors
Frameshift suppressors are mutant tRNA molecules that
have a 4 base anticodon rather than the normal 3 base
anticodon.
This re-orients the reading frame properly and allows the
protein to be made.
normal
AAA CCC GGG
mutant
AAA XCC CGG G
Suppressed AAA XCCC GGG

UV Light as a Mutagen
Ultraviolet (UV) light can cause mutations in a genetic
sequence.
This requires that there are two Thymines adjacent to one
another in the sequence.
How rare of an event is adjacent Thymines?
There are only four nucleotides in a DNA molecule so
adjacent Thymines are very common.

UV Light as a Mutagen
When adjacent Thymines are irradiated with UV light,
they form a Thymine dimer where the normal hydrogen
bonds which usually interact with its complimentary bases
are replaced with covalent bonds between Thymines
creating a Thymine Dimer.
Thymine Dimers prevent DNA replication or Transcription
of the gene into mRNA.
The cell recognizes this as a problem and can intitiate
Excision Repair (Dark Repair) or Light Repair.

UV Light as a Mutagen
Watch the following video by right clicking on the link and
opening the hyperlink.
http://
highered.mcgraw-hill.com/olc/dl/120082/micro18.swf

UV Light as a Mutagen
The dimer and the surrounding 10 13 bases are
excised out during excision repair and the opposite strand
of DNA is used as template to resynthesize the lost
bases.
The DNA polymerase used to resynthesize the strand is
less faithful than the DNA polymerases used in S phase
of the cell cycle.
The Polymerase makes mistakes anywhere along the
excised region and can insert the wrong base causing a
single base substitution which is an inheritable change in
the genetic code.

X1687-12B Yeast Strain

In lab, well be using a yeast strain that is known to be a
multiple auxotroph and cannot synthesize its own
tryptophan, lysine, or histidine.
Well use UV light as a mutagen and attempt to suppress
these mutations and reverse their effects.
If this strain can synthesize its own tryptophan, lysine, or
histidine, it will have been suppressed.

X1687-12B Yeast Strain

We might produce one of two types of suppressors.
Intragenic Suppression
Extragenic Suppression

The intragenic suppression is a mutation event that

occurs within the gene that is causing the mutant
phenotype.
Extragenic Suppression will involve a mutation event in
another gene completely separate from the gene causing
the mutant phenotype.

X1687-12B Yeast Strain

How will we know which type of mutation has occurred?
Well use some simple statistics.
Intracodon Suppression (also called a Reversion to wild
type) occurs in about 1 in 106 cells when using UV light as
a mutagen.
Lets review Intracodon Suppression.

Intracodon Suppression

In the wild type, youll see that Arginine is the amino acid
that gives proper folding of the protein.
After mutation, Serine is placed in the protein where
arginine previously was causing protein mis-folding and a
mutant phenotype.

Intracodon Suppression

A further base substitution occurs within (Intra) the codon

giving the wild-type phenotype.
In the first example, Arginine is placed back in the amino
acid sequence. The second example will substitute
Asparagine which is similar in structure to Arginine.
In either case, the protein will fold properly and give the
wild-type phenotype.

X1687-12B Yeast Strain

UV light initiates excision repair which causes mutations.
If this mutation occurs at the mutant codon the strain of
yeast can be reverted back to wild type.
Again, there is about a one in a million chance of this
happening.
Are those too long of odds to see in the real world?
No, remember that a single milliliter of culture can contain
several hundred million cells.

X1687-12B Yeast Strain

This yeast strain is a triple auxotroph requiring Trp, Lys,
and His supplements in media to grow.
After UV exposure, this yeast strain will become
prototrophic for all 3 and are now able to grow on media
that is lacking the supplements.
This fact will allow us to determine whether our
suppressor mutation was Intragenic or Extragenic.

X1687-12B Yeast Strain

Suppose the genetic make up of this strain is as shown
below.

We see that each gene encodes a premature nonsense

codon in each of its mRNAs causing truncated proteins
which will not fold properly.

X1687-12B Yeast Strain

Looking just at the Trp mRNA, we know that intracodon
suppression (reversion) can occur at a rate of about one
in a million.

If this occurred, then the yeast strain would incorporate

Serine at the spot previously causing truncated proteins.

X1687-12B Yeast Strain

We can make the exact same argument for see a
reversion in His or Lys pathways.
The chances of an individual intracodon suppressor
(reversion) in either of these pathways is the same 1 in a
million.
How does your argument change if you see all three
pathways corrected simultaneously in the same cell?

X1687-12B Yeast Strain

You could argue that 3 simultaneous intracodon
suppressions (reversions) have occurred.

What are the chances of that happening?

X1687-12B Yeast Strain

One reversion is about a 1 in 106 chance.
If you want to predict the odds of two events, multiply the
odds of each individual event together.
Having two simultaneous reversions would be a one in
1012 occurrence.
Having three simultaneous reversions would be a one in
1018 occurrence.
Do you really want to argue a 10 18 occurrence?

X1687-12B Yeast Strain

Extragenic suppression is an alternative argument.
Extragenic suppression is the mutation of another gene
that reverses the first mutation.
Here, recognize that the mutations in all three pathways
were the same nonsense codon leading to truncations.

X1687-12B Yeast Strain

What other gene might be mutated to produce the wild
type phenotype for all three traits?
Nonsense suppressors can suppress nonsense codons
by mutating a gene which encodes tRNAs.
A one time mutation event using UV light can generate a
tRNA that has an anticodon capable of reading UAA.
This event can occur at a rate of about 1 in 10 6 cells.

X1687-12B Yeast Strain

Would a single mutation event creating a tRNA with a
AUU anticodon allow full length and functional proteins
from each of these mRNAs?
Yes, the yeast strain can now make its own Trp, His, and
Lys.

X1687-12B Yeast Strain

What is the best argument?
We observed 3 simultaneous intracodon suppressions
(reversions) at odds of 1 in 10 18?
We observed one extragenic nonsense suppressor
mutations at odds of 1 in 106?
Statistics requires that we go with later.
We have observe an extragenic nonsense suppressor.