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The mutation in DNA sequences with dyad symmetry upstream of the puc promoter affects oxygen regulation of the puc operon(s) in Rhodobacter sphaeroides

Wu Yongqiang, Kaplan S

Shanghai Institute of Plant Physiology, The Chinese Academy of Sciences, Shanghai 200032

Abstract

Seven mutations and five 10-bp deletions were constructed in the 5^ noncoding region of the puc operon by in vitro manipulation of cloned puc upstream sequence with a modified Polymerase Chain Reaction (PCR) and oligonucleotide-directed mutagenesis.Transcriptional fusions of the puc upstream region containing different cis-mutations or 10-bp deletions were constructed by using the reporter molecule encoded by lacZ.The role of the target site controlling puc expression was investigated by analysing the effects of the mutations and deletions on the #bata#-galactosidase (#bata#-gal) activity resulting from the fusions when present in wild type R. sphaeroides 241 grown chemoheterotrophically and fermentatively. #bata#-gal activity of PCF611 with A to G transition mutation at-12 within a region of dyad symmetry was about 7.8 foldthat of the wild type fusion construct in the presence of O_2.The puc upstream sequence of PCFK carrying three mutations at -197,-193 and 190 between positions positions-199 to -185 within another dyad symmetry GCAGCN_5GCTGC produced a #bata#-gal activity 6.8 times as high as wild type activity.Binding of IHF is believed to induce a DNA bend which was proposed to faciliated interation between the O_(2)-regulatory repressor and the two regions of dyad symmetry.All these fusions showed almost the same level of lacZ gene expression under anaerobic condition.

Key words: Oxygen regulation;Puc operon;Dyad symmetry;Mutation

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Citing This Article：

Wu Yongqiang, Kaplan S. The mutation in DNA sequences with dyad symmetry upstream of the puc promoter affects oxygen regulation of the puc operon(s) in Rhodobacter sphaeroides. Acta Physiol Sin 1996; 48 (2): (in Chinese with English abstract).