Escherichia coli rpiA gene encoding ribose phosphate isomerase A.
The rpiA gene encoding ribose phosphate isomerase A was cloned from phage 1A2(471) of the Kohara gene library. Subcloning, restriction, and complementation analyses revealed an 1,800-bp SspI-generated DNA fragment that contained the entire control and coding sequences. This DNA fragment was sequenced and shown to harbor an open reading frame of 219 codons, sufficient to encode a polypeptide with an M(r) of 22,845. The synthesis of the rpiA-encoded polypeptide was detected by analysis of minicells, which established the subunit M(r) as 27,000. The assignment of the correct reading frame was confirmed by amino-terminal analysis of partially purified ribose phosphate isomerase A. Our data indicate that the enzyme is composed of two identical subunits. The 5' end of the rpiA-specified transcript was analyzed by primer extension, which revealed a well-conserved -10 region 34 bp upstream of the presumed translation start codon. Analysis of the 3' end of the transcript by S1 nuclease mapping showed that transcription termination occurred within an adenylate-rich sequence following a guanylate-cytidylate-rich stem-loop structure resembling a rho factor-independent transcription terminator. Host strains harboring the rpiA gene in a multicopy plasmid contained up to 42-fold as much ribose phosphate isomerase A activity as the haploid strain.
Hove-Jensen
B
B
Department of Biological Chemistry, University Institute of Molecular Biology, Copenhagen K, Denmark.
Maigaard
M
M
D019747:Aldose-Ketose Isomerases; D000595:Amino Acid Sequence; D001483:Base Sequence; D002238:Carbohydrate Epimerases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; C022548:ribosephosphate isomerase