Abstract
Phosphoribosylpyrophosphate (P-Rib-PP) synthetase of Escherichia coli has been purified to near homogeneity from a strain harboring the prs gene, encoding P-Rib-PP synthetase, on a multicopy plasmid. Analysis of the enzyme showed that it required inorganic phosphate for activity and for stability. Magnesium ions were required both as a complex with the substrate ATP and as a free cation. P-Rib-PP synthetase activity was inhibited strongly by ADP. Kinetic analysis indicated multiple sites of action of ADP. In addition apparent substrate inhibition was exerted by ribose 5-phosphate in the presence of ADP. The nucleotide sequence of the E. coli prs gene has been determined and the coding segment established. The deduced amino acid sequence of P-Rib-PP synthetase contained 314 amino acid residues and the molecular weight was calculated as 34,060. The initiation site of transcription was determined. This site was preceded by well conserved -10 and -35 consensus sequences (pdT-dA-dG-dA-dA-dT and pdT-dT-dG-dA-dT-dG, respectively). The transcription initiation site preceded the potential translation initiation site by 302 nucleotides. Transcription terminated approximately 35 nucleotides downstream from the UAA translation stop codon, within a Thy-rich region following an inverted repeat sequence, indicative of an rho-independent transcription terminator.
Highlights
From the $EnzymeDiuision, Institute of Biological Chemistry B, Uniuersity of Copenhagen, Copenhagen, Denmark and the TDepartment of Biochemistry, University of Illinois, Urbana, Illinais 61802
Properties of E. coli P-Rib-PP Synthetase-P-Rib-PP synthetase was readily purified to near homogeneity in high yield from E. coli H0561 cells
Analysis of the amino acids released from P-Rib-PP synthetase by a mixture of carboxypeptidases A and B indicated that the carboxyl terminus of both the E. coli and S. typhimurium enzymes was -Glu-His-COOH
Summary
Properties of E. coli P-Rib-PP Synthetase-P-Rib-PP synthetase was readily purified to near homogeneity in high yield from E. coli H0561 cells. Identification of the prs Promoter-Seven nucleotide pairs upstream of the identified transcription initiation site is the sequence pdT-dA-dG-dA-dA-dT (nucleotides 201-206, Fig. 4), which is a well conserved -10 region. Translation of prs-Native P-Rib-PP synthetase contains proline as theamino-terminal amino acid residue This amino acid is coded for by the CCTproline codon (nucleotides 518520,Fig. 4). Seven nucleotide pairs further upstream is the sequence pdG-dA-dG-dG-dT (nucleotides 503-507,Fig. 4) This sequence is complementary to parts of the 3‘ end of 16 S rRNA and thusmay function as a ribosome binding site (Shine andDalgarno, 1974).These data suggest that one methionyl residue is hydrolyzed from the primary P-Rib-PP synthetase translation product to obtain the native form. 214 amino acid residues of the Salmonella enzyme have been determined to date by automated Edman degradation of the enzyme and peptides derived from cyanogen bromide degradation? Only 4 of these residues differ from the amino acid
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