Topic C - Mutation, Reversion and Suppression

Naming a Bacterial Gene

1. Three lowercase italic letters refer to the gene’s function

• Example: his = gene involved in histidine biosynthesis

2. More than one gene involved in a process → followed up with an upper case italic letter to distinguish between different genes

• Example: hisA, hisB, hisC are different genes involved in histidine biosynthesis

3. Protein product has no italics, referred to with capital letter

• Example: HisA

Naming a Mutation

1. Mutation that inactives the product: Superscript minus

2. Wildtype gene (functional product): Superscript plus

3. Gain of Function → More plus signs (hisA++) or asterisk (hisA*)

4. Many different mutations in a single gene:

• Different alleles are given a number

• Example: hisA4 → mutation #4 in the hisA gene

Special Notation in Mutaitons

Deletions = Δ

Insertion = ::

Example: (delta)hisA = hisA gene has been deleted

Example2: hisA::Kan®

-Insertion of Kanamycin resistance gene in hisA

Mutation and Premutation

Heritable changes in the DNA sequence

-Premutation - result of DNA damage, if not corrected before replication it develops into a mutation

Small-Scale Mutations - Base Substitution

Change of one base pair to another, can have multiple base substitutions (single, double, triple, etc)

-Are classified by effect on protein

Silent - Altered codon encodes same amino acid, no effect on protein

Missense - Changes a codon to encode a different amino acid

-may alter protein structure/function

-conservative vs non-conservative

Nonsense - Introduces a premature stop codon

-Results in a truncated protein, effect depends on location

Small-Scale Mutations - Frameshift Mutations

Insertion or deletion of one or more base pairs (indel) resulting in a shift of reading frame

-Unless multiples of three

-Ribosome cannot read codons correctly, amino acids in sequence are now different

Effects the entire sequences of amino acids after the mutation, often produces a truncated protein

Large-Scale Mutation

Deletion - Removes all or part of a gene
-Often results in loss of function phenotype

Insertion - Adds DNA not present before

-Often results in loss of function if inserted between gene and promoter, or new gene expression if insertion has RBS, start/stop codon

Inversion - Segment of DNA becomes inverted

-Causes all genes to face in opposite direction

-Often results in loss of function since the mRNA is flipped (Ribosome unable to bind since RBS is at the 3’ end) or the orientation of the promoter is changed (nothing changes in gene expression)

Translocation - Segment of DNA moved to different location in chromosome

-Can result in loss of function (if gene moved away from promoter) or no change in gene expression if gene moved towards the promoter

Reversion and Revertant

Reversion - Process in which a mutant regains the wildtype phenotype

-May be spontaneous or induced mutation

Revertant - Mutant that has undergone reversion to the wildtype phenotype

Mutations Causing Reversion

True Reversion/Back Mutation - Mutation restores wildtype DNA sequence, or

Suppression (Pseudo-Reversion) - Additional mutation at second site restores wildtype phenotype

-Second mutation suppresses original mutation, but original mutation still exists in DNA sequence

Reversion Frequency

Frequency of cells in a population that have reverted to the wildtype phenotype

RofF = Number of Revertant / Total number of mutant bacteria

-Rates are lower than mutation rates (require more specific sequence change to occur)

-Probability of true reversion is very low, requires the most specific change

-Observed reversion frequency is much higher, therefore most reversion is due to suppression

Suppressor Mutation

Mutation in DNA relieves the effect of another mutation

-Divided into two types: Intragenic and Intergenic Suppression

Intragenic Suppression

Reversions that occur due to mutations in the same gene as the original mutation

Intergenic Suppression

Mutation in a second gene suppresses the phenotype of the mutation in the original gene

Mechanisms of Intragenic Suppression

1. Base substitution

• Can be same or different codon reversion

1. Frameshift mutation

Base Substitution

Same Codon Reversion

-Original mutation in the same codon is mutated, resulting in the mutant phenotype being suppressed (WT phenotype now expressed)

Different Codon Reversion

-Second mutation in a different codon results in the WT phenotype being expressed

Frameshift Mutations

Second frameshift mutation within same gene as the original mutation resulting in suppression
1. Second mutation must be near original mutation

-As length of frame-shifted sequence increases, so does:

• Probability of encountering stop codon

• Probability of irreversibly disrupting protein function

2. Altered region of protein must be able to withstand change in sequence

-Frame-shifted region will have completely different sequence from wildtype

-Region can’t be essential for function

Mechanisms of Intergenic Suppression

1. Interaction suppressors

2. Bypass suppressors

3. Informational suppressors

Interaction Suppressors

Suppresses a mutation that disrupts protein/protein interaction

-First mutation blocks interaction

-Second mutation restores protein interaction

Based on amino acid characteristics (charge, size, etc)

Bypass Suppressor

Turns on a new pathway that eliminates the need for the mutant gene

-Turns on 2nd pathway that was turned off by mutation #1

-Most common type of intergenic suppressor

Informational Suppressor

Alter cell’s translation machinery so that the original mutation is misread

-Functional protein made from mutant gene

Mutation in tRNA anticodon recognizes stop codon caused by original nonsense mutation

-Nonsense stop codon is translated into an amino acid by the mutated tRNA

-Full length protein produced, wildtype phenotype achieved

Informational Suppressor Example

OG mutation is nonsense mutation

-introduces premature stop codon in gene, resulting in truncated protein

-produces mutant phenotype in wildtype bacterial cells

Suppressor mutation is in a tRNA gene

-Nonsense stop codon is translated into an amino acid codon by the mutated tRNA

-Full length protein is produced

-Wildtype phenotype seen in strain with suppressor mutation