PMID:8366047
| Citation |
Hove-Jensen, B and Maigaard, M (1993) Escherichia coli rpiA gene encoding ribose phosphate isomerase A. J. Bacteriol. 175:5628-35 |
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| Abstract |
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. |
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| Keywords |
Aldose-Ketose Isomerases; Amino Acid Sequence; Base Sequence; Carbohydrate Epimerases/biosynthesis; Carbohydrate Epimerases/genetics; Carbohydrate Epimerases/metabolism; Cloning, Molecular; DNA, Bacterial; Escherichia coli/enzymology; Escherichia coli/genetics; Gene Expression Regulation, Bacterial; Genes, Bacterial; Molecular Sequence Data; Plasmids; Promoter Regions, Genetic; Transcription, Genetic |
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