| Abstract: | The Escherichia coli gene purF, coding for 5-phosphoribosylamine:glutamine pyrophosphate phosphoribosyltransferase (amidophosphoribosyltransferase) was subcloned from a ColE1-purF plasmid into pBR322. Amidophosphoribosyltransferase levels were elevated more than 5-fold in the ColE1-purF plasmid-bearing strain compared to the wild type control, and a further 10- to 13-fold elevation was observed in several pBR322 derivatives. The nucleotide sequence of a 2478-base pair PvuI-HinfI fragment encoding purF was determined. The purF45 structural gene codes for a 56,395 Mr protein chain having 504 amino acid residues. Methionine-1 is removed by processing in vivo leaving cysteine as the NH2-terminal residue. The deduced amino acid sequence was confirmed by comparisons with the NH2-terminal amino acid sequence determined by automated Edman degradation (Tso, J. Y., Hermodson, M. A., and Zalkin, H. (1982) J. Biol. Chem. 257, 3532-3536) and amino acid analyses of CNBr peptides including a 4-residue peptide from the CO2H terminus of the enzyme. Nucleotide sequences characteristic of bacterial promoter-operator regions were identified in the 5' flanking region. The coding region appears to be preceded by a 277-297 nucleotide mRNA leader. A deletion removing the putative promoter-operator region results in defective purF expression. |
