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

J Mol Biol. 1983 Aug 5;168(2):285-305. doi: 10.1016/s0022-2836(83)80019-9.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Cloning, Molecular*
  • DNA Restriction Enzymes
  • DNA, Bacterial
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / enzymology
  • Escherichia coli / genetics*
  • Gene Expression Regulation
  • Genes, Bacterial*
  • Mutation
  • Operon
  • Phosphofructokinase-1 / genetics*
  • Phosphofructokinase-1 / metabolism
  • Plasmids
  • Transcription, Genetic

Substances

  • DNA, Bacterial
  • Phosphofructokinase-1
  • DNA Restriction Enzymes

Associated data

  • GENBANK/K00128