ESCHERICHIA-COLI RPIA GENE ENCODING RIBOSE PHOSPHATE ISOMERASE-A

被引:54
作者
HOVEJENSEN, B
MAIGAARD, M
机构
关键词
D O I
10.1128/JB.175.17.5628-5635.1993
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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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页码:5628 / 5635
页数:8
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