Pseudo-reversion: Intragenic suppressors

Intragenic suppressors are second mutations within the same gene that restore function of the mutant gene product.

For example -

  • Base substitution mutations:

    1. same codon / same nucleotide:
         UGG -> UGA -> UGC 
      trp stop cys

    2. same codon / different nucleotide:
         UGG -> UGA -> CGA
      trp stop arg

    3. different codon in the same gene:
      arg glu his leu glu his leu glu leu

      (wild-type) (1 mutant) (pseudo-revertant)
  • Frameshift mutations:

       5' AUG UGG GGA CCC AAG GGU AGC CCC ... 3'	(wild-type)
    met trp gly pro lys gly ser pro ...

    5' AUG UGG GGG ACC CAA GGG UAG CCC C.. 3' (1 mutant)
    met trp gly thr gln gly stop

    5' AUG UGG GGG ACC AAG GGU AGC CCC ... 3' (pseudo-revertant)
    met trp gly thr lys gly ser pro ...

    (Different ways of repairing an error in sentence structure provide a nice analogy for the effects of mutations on the resulting sequence. For examples, see the following example contributed by Deanna Raineri.)
  • Intragenic suppressors that affect protein structure (allele specific):

    Example: Tryptophan synthetase encodes an enzyme required for the biosynthesis of the amino acid tryptophan in E. coli [Helinsky and Yanofsky].

            mutation	reversion	         backcross
    Gly210 -------> Glu210 ---------> Glu210 Cys174 ---------> Gly210 Cys174
    (active) (inactive) (active) (inactive)

    (If the amino acid is not shown, it is assumed to be wild-type.)

    Note amino acid substitution at position 174 is only active if the amino acid at position 210 is also changed, not if the wild type amino acid is at position 210 -- that is, the amino acid substition that restores activity in the revertant is specific for the allele at position 210. These results suggest that amino acid at position 210 interacts with amino acid at position 174

    Another experiment:

          mutant            revertant
    Leu176 Val212 ---> Arg176 Val212
    (inactive) (active)

    This result further suggests that amino acids at positions 210 and 212 interact with amino acids at positions 174 and 176. The proximity of these amino acids can be seen in the crystal structure of TrpA protein. In Figure A below the amino acids are highlighted in red in a secondary structure diagram, and in Figure B a close up of the amino acids is shown.



  • Intragenic suppressors that affect protein function (not allele specific):

    Example: Tryptophan repressor (encoded by the trpR gene) regulates expression of tryptophan biosynthetic genes [Klig, Oxender, and Yanofsky].

    Low [Tryptophan] = TrpR cannot bind DNA, no repression, Trp biosynthesis turned ON
    High [Tryptophan] = Trp binds to TrpR protein, Trp-TrpR complex binds DNA, repression occurs, Trp biosynthesis turned OFF.

    Which amino acids in the TrpR protein recognize DNA? Approach - look for pseudorevertants with increased DNA binding. Select for revertants of 5 different trpR alleles (missense mutants) that were defective for DNA binding. (This phenotype might either be due to amino acid changes that prevent Trp binding to the TrpR protein or amino acid changes that prevent protein-DNA interactions.)

    Predicted types of revertants:

    1. Allele specific suppressors due to interactions between mutant amino acids and the nucleotide sequence

    2. Non-allele specific suppressors due to indirect effects on protein function
      - mutations that increase tryptophan binding or specific DNA binding
      - mutations that increase nonspecific DNA binding
      - mutations that increase protein stability resulting in higher [protein]

    Types of mutants may be distinguished by biochemical and genetic approaches
    - Biochemical approach = assay each separate activity
    - Genetic approach = determine if revertant is allele specific


                   mutation             reversion            backcross
          GAG   ACG ------->  GAG   ATG  -------> AAG    ATG  ------->  AAG   ACG
         Glu18 Thr44         Glu18 Met44         Lys18  Met44          Lys18 Thr44
         (active)	         (inactive)           (active)	             (active)

    Note that the Lys18 mutation works with both a Thr or Met at position 44. These results demonstrate that it is not an allele specific revertant. Furthermore the Lys18 mutation gives the protein a super-repressor phenotype (that is, it has more DNA-binding activity than the wild-type) protein. This result indicates that the reversion is due to increased activity of the protein, not interactions between amino acid in the original mutant and the amino acid in the revertant. These amino acid residues are distant from each other in the crystal structure of the TrpR protein as shown in the Figure below.

    This amino acid was not previously predicted to play a role in DNA-binding by the TrpR protein based upon biochemical studies, demonstrating the value of a genetic approach even if a protein has been thoroughly studied biochemically.

    An aside: "You get what you select for." Note that these super-repressor mutants were isolated by selecting for increased DNA-binding by missense mutants that were deficient for this activity. This approach does not work well if you start with the wild-type trpR because the wild-type protein binds DNA so well that it would be difficult to detect the "increased DNA-binding phenotype" without doing brute-force biochemical assays on the mutants. The take-home point is that sometimes you have to devise sneaky schemes to isolate interesting, informative types of mutants. (For a nice discussion of the pearls and pitfalls of suppressor analysis, see [Manson, M. 2000. Allele-specific suppression as a tool to study protein-protein interactions in bacteria. Methods 20: 18-34]).

    Return to Mcbio 316 supplement.

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