Intragenic suppressors are second mutations within the same gene that restore function of the mutant gene product.
For example -
UGG -> UGA -> UGC
trp stop cys
UGG -> UGA -> CGA
trp stop arg
CGA GAG CAU -> CUA GAG CAU -> CUA GAG CCU
arg glu his leu glu his leu glu leu
(wild-type) (1° mutant) (pseudo-revertant)
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.)
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
Leu176 Val212 ---> Arg176 Val212
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.
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:
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]).
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