Characterization of the pleiotropic LysR-type transcription regulator LeuO of Escherichia coli

Nucleic Acids Res. 2019 Aug 22;47(14):7363-7379. doi: 10.1093/nar/gkz506.

Abstract

LeuO is a pleiotropic LysR-type transcriptional regulator (LTTR) and co-regulator of the abundant nucleoid-associated repressor protein H-NS in Gammaproteobacteria. As other LTTRs, LeuO is a tetramer that is formed by dimerization of the N-terminal DNA-binding domain (DBD) and C-terminal effector-binding domain (EBD). To characterize the Escherichia coli LeuO protein, we screened for LeuO mutants that activate the cas (CRISPR-associated/Cascade) promoter more effectively than wild-type LeuO. This yielded nine mutants carrying amino acid substitutions in the dimerization interface of the regulatory EBD, as shown by solving the EBD's crystal structure. Superimposing of the crystal structures of LeuO-EBD and LeuO-S120D-EBD suggests that the Ser120 to Asp substitution triggers a structural change that is related to effector-induced structural changes of LTTRs. Corresponding functional analyses demonstrated that LeuO-S120D has a higher DNA-binding affinity than wild-type LeuO. Further, a palindromic DNA-binding core-site and a consensus sequence were identified by DNase I footprinting with LeuO-S120D as well as with the dimeric DBD. The data suggest that LeuO-S120D mimics an effector-induced form of LeuO regulating a distinct set of target loci. In general, constitutive mutants and determining the DNA-binding specificity of the DBD-dimer are feasible approaches to characterize LTTRs of unknown function.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA / chemistry
  • DNA / genetics
  • DNA / metabolism
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / genetics*
  • Escherichia coli Proteins / metabolism
  • Gene Expression Regulation, Bacterial*
  • Genetic Pleiotropy / genetics*
  • Models, Molecular
  • Mutation
  • Nucleic Acid Conformation
  • Promoter Regions, Genetic / genetics*
  • Protein Binding
  • Protein Domains
  • Protein Multimerization
  • Transcription Factors / chemistry
  • Transcription Factors / genetics*
  • Transcription Factors / metabolism

Substances

  • DNA-Binding Proteins
  • Escherichia coli Proteins
  • LeuO protein, E coli
  • Transcription Factors
  • DNA