THE DEFECTIVE PHOSPHORIBOSYL DIPHOSPHATE SYNTHASE IN A TEMPERATURE-SENSITIVE PRS-2 MUTANT OF ESCHERICHIA-COLI IS COMPENSATED BY INCREASED ENZYME-SYNTHESIS

An Escherichia coil strain which is temperature-sensitive for growth d ue to a mutation (prs-2) causing a defective phosphoribosyl diphosphat e (PRPP) synthase has been characterired. The temperature-sensitive mu tation was mapped to a 276 bp HindIII-BssHII DNA fragment located with in the open reading frame specifying the PRPP synthase polypeptide. Cl oning and sequencing of the mutant allele revealed two mutations. One, a G --> A transition, located in the ninth codon, was responsible for the temperature conditional phenotype and resulted in a serine residu e at this position. The wild-type codon at this position specified a g lycine residue that is conserved among PRPP synthases across a broad p hylogenetic range. Cells harbouring the glycine-to-serine alteration s pecified by a plasmid contained approximately 50% of the PRPP synthase activity of cells harbouring a plasmid-borne wildtype allele, both gr own at 25 degrees C. The mutant enzyme had nearly normal heat stabilit y, as long as it was synthesized at 25 degrees C. In contrast, there w as hardly any PRPP synthase activity or anti-PRPP synthase antibody cr oss-reactive material present in cells harbouring the glycine to serin e alteration following temperature shift to 42 degrees C. The other mu tation was a C --> T transition located 39 bp upstream of the G --> A mutation, i.e. outside the coding sequence and close to the Shine-Dalg arno sequence. Cells harbouring only the C --> T mutation in a plasmid contained approximately three times as much PRPP synthase activity as a strain harbouring a plasmid-borne wild-type prs allele. In cells ha rbouring both mutations, the C --> T mutation appeared to compensate f or the C --> A mutation by increasing the amount of a partially defect ive enzyme at the permissive temperature.