Biomedical subjects

Roy David Magnuson

Publications and source records attributed to Roy David Magnuson.

3 recordsLinked to original sources

PubMed2005

Characterization of the Phd repressor-antitoxin boundary.

The P1 plasmid addiction operon (a classic toxin-antitoxin system) encodes Phd, an unstable 73-amino-acid repressor-antitoxin protein, and Doc, a stable toxin. It was previously shown by deletion analysis that the N terminus of Phd was required for repressor activity and that the C terminus was required for antitoxin activity. Since only a quarter of the protein or less was required for both activities, it was hypothesized that Phd might have a modular organization. To further test the modular hypothesis, we constructed and characterized a set of 30 point mutations in the third and fourth quarters of Phd. Four mutations (PhdA36H, V37A, I38A, and F44A) had major defects in repressor activity. Five mutations (PhdD53A, D53R, E55A, F56A, and F60A) had major defects in antitoxin activity. As predicted by the modular hypothesis, point mutations affecting each activity belonged to disjoint, rather than overlapping, sets and were separated rather than interspersed within the linear sequence. A final deletion experiment demonstrated that the C-terminal 24 amino acid residues of Phd (preceded by a methionine) retained full antitoxin activity.

Amino Acid Substitution↗

PubMed2005

Percolation of the phd repressor-operator interface.

Transcription of the P1 plasmid addiction operon, a prototypical toxin-antitoxin system, is negatively autoregulated by the products of the operon. The Phd repressor-antitoxin protein binds to 8-bp palindromic Phd-binding sites in the promoter region and thereby represses transcription. The toxin, Doc, mediates cooperative interactions between adjacent Phd-binding sites and thereby enhances repression. Here, we describe a homologous operon from Salmonella enterica serovar Typhimurium which has the same pattern of regulation but an altered repressor-operator specificity. This difference in specificity maps to the seventh amino acid of the repressor and to the symmetric first and eighth positions of the corresponding palindromic repressor-binding sites. Thus, the repressor-operator interface has coevolved so as to retain the interaction while altering the specificity. Within an alignment of homologous repressors, the seventh amino acid of the repressor is highly variable, indicating that evolutionary changes in repressor specificity may be common in this protein family. We suggest that the robust properties of the negative feedback loop, the fuzzy recognition in the operator-repressor interface, and the duplication and divergence of the repressor-binding sites have facilitated the speciation of this repressor-operator interface. These three features may allow the repressor-operator system to percolate within a nearly neutral network of single-step mutations without the necessity of invoking simultaneous mutations, low-fitness intermediates, or other improbable or rate-limiting mechanisms.

Amino Acid Sequence↗

PubMed2004

Modular organization of the Phd repressor/antitoxin protein.

The P1 plasmid addiction operon is a compact genetic structure consisting of promoter, operator, antitoxin gene (phd), and toxin gene (doc). The 73-amino-acid antitoxin protein, Phd, has two distinct functions: it represses transcription (by binding to its operator) and it prevents host death (by binding and neutralizing the toxin). Here, we show that the N terminus of Phd is required for repressor but not antitoxin activity. Conversely, the C terminus is required for antitoxin but not repressor activity. Only a quarter of the protein, the resolution limit of this analysis, was required for both activities. We suggest that the plasmid addiction operon is a composite of two evolutionarily separable modules, an operator-repressor module and an antitoxin-toxin module. Consideration of similar antitoxin proteins and their surroundings indicates that modular exchange may contribute to antitoxin and operon diversity.

Amino Acid Sequence↗