Expression and purification of the cynR regulatory gene product: CynR is a DNA-binding protein

J Bacteriol. 1993 Dec;175(24):7990-9. doi: 10.1128/jb.175.24.7990-7999.1993.

Abstract

The CynR protein, a member of the LysR family, positively regulates the Escherichia coli cyn operon and negatively autoregulates its own transcription. By S1 mapping analysis, the in vivo cynR transcription start site was located 63 bp upstream of the cynTSX operon transcription start site. Topologically, the cynR and cynTSX promoters overlap and direct transcription in opposite directions. The CynR translation initiation codon was identified by oligonucleotide-directed mutagenesis, and the CynR coding sequence was cloned under the control of a T7 phage promoter. The CynR protein was stably expressed at a high level with a T7 RNA polymerase-T7 phage promoter system. Purification by ion-exchange chromatography, affinity chromatography, and ammonium sulfate fractionation yielded pure CynR protein. Gel shift assays confirmed that CynR is a DNA-binding protein like the other members of the LysR family. The CynR regulatory protein binds specifically to a 136-bp DNA fragment encompassing both the cynR and the cynTSX promoters.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / biosynthesis*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / isolation & purification
  • Base Sequence
  • DNA-Binding Proteins / biosynthesis*
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism*
  • Escherichia coli Proteins*
  • Gene Expression
  • Gene Expression Regulation, Bacterial*
  • Genes, Bacterial*
  • Genes, Regulator*
  • Introns
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Oligodeoxyribonucleotides
  • Operon*
  • Plasmids
  • Promoter Regions, Genetic
  • RNA, Messenger / biosynthesis
  • RNA, Messenger / metabolism
  • Restriction Mapping
  • Trans-Activators*
  • Transcription Factors / biosynthesis*
  • Transcription Factors / genetics
  • Transcription Factors / isolation & purification
  • Transcription, Genetic

Substances

  • Bacterial Proteins
  • DNA-Binding Proteins
  • Escherichia coli Proteins
  • Oligodeoxyribonucleotides
  • RNA, Messenger
  • Trans-Activators
  • Transcription Factors
  • cynR protein, E coli