The positive screening procedure previously described was used in order to select, clone and characterize mutants defective in negative feedback control by UTP of the yeast carbamoylphosphate synthetase-aspartate transcarbamylase protein (CPSase-ATCase). The selection procedure was improved by adding a general mapping method for dominant mutations in order to avoid sequencing the whole URA2 allele (7 kb). All 16 mutants obtained carry missense mutations leading to single amino acid replacements: five of them are located in the CPSase domain while the other 11 are in the ATCase domain. In these 16 mutants, ATCase is no longer inhibited by UTP although CPSase retains full sensitivity to the effector, suggesting that the regulation of the two activities involve distinct mechanisms. Amino acid replacements in the ATCase domain were located on a three-dimensional model structure of the yeast ATCase domain. They are clustered in two regions of this domain which must be directly involved in the feedback process.

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Journal Article | Research Support, Non-U.S. Gov't

Authors

Jaquet L, Serre V, Lollier M, Penverne B, Hervé G, Souciet JL, Potier S

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