Molecular cloning of the gene for phosphofructokinase-2 of Escherichia coli and the nature of a mutation, pfkB1, causing a high level of the enzyme.
The pfkB gene of Escherichia coli is known to specify a minor phosphofructokinase, Pfk-2, in the wild-type strain; the pfkB1 mutation causes a 25-fold increase in the amount of Pfk-2 so that it adequately substitutes for mutational loss of the major phosphofructokinase, Pfk-1 (specified by pfkA); and another closely linked mutation, pfkB10, affects the structure of Pfk-2. This paper is about the pfkB1 mutation. pfkB+, pfkB1 and pfkB1 pfkB10 were cloned and subcloned on plasmid pBR322; their functions were carried, in all three cases, by a 2.1 X 10(3) base-pair fragment with a similar or identical restriction pattern. Experiments with "maxicells" confirmed that the cloned fragments included the structural gene as well as the determinants of its level of expression. Results of N-terminal sequencing of the enzyme matched with the DNA sequence and established the position and direction of the gene. A HindIII-SmaI fragment of 408 base-pairs, which included 294 base-pairs of the non-coding 5' region and 114 base-pairs of the protein coding sequence, was fused to galK on the promoter cloning vector pK01; in the fusion pfkB1 caused a high level expression of galK. Transcription in vitro from pfkB+ and pfkB1 allowed the determination of the +1 position in both cases, at about 19 base-pairs before the initiating methionine codon; the level of transcription was much higher from pfkB1 than from pfkB+. The DNA sequence of the 408 base-pair fragments from pfkB+ and pfkB1 were found to differ only in a single residue, the pfkB1 mutation thus proving to be a C to T change at position about -12 from the initiation of transcription.
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D009154:Mutation; D009876:Operon; D010732:Phosphofructokinase-1 / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D010732:Phosphofructokinase-1; D004262:DNA Restriction Enzymes