Differential protein-DNA contacts for activation and repression by ArgP, a LysR-type (LTTR) transcriptional regulator in Escherichia coli

Microbiol Res. 2018 Jan;206:141-158. doi: 10.1016/j.micres.2017.10.009. Epub 2017 Oct 23.

Abstract

ArgP is a LysR-type transcriptional regulator (LTTR) that operates with two effector molecules, lysine and arginine, to differentially regulate gene expression. Effector-free ArgP stimulates transcription of all investigated regulon members, except argO, whereas lysine abolishes this effect. Activation of argO, encoding an exporter for arginine and canavanine, is strictly dependent on arginine-bound ArgP. Lysine counteracts this effect and even though lysine-bound ArgP stimulates RNA polymerase recruitment at the argO promoter, the complex is non-productive. It is presently unclear what distinguishes argO from other ArgP targets and how binding of arginine and lysine translates in antagonistic effects on promoter activity. Here we generate high resolution contact maps of effector-free and effector-bound ArgP-DNA interactions and identify the sequence 5'-CTTAT as the consensus recognition motif for ArgP binding. argO is the only operator at which ArgP binding overlaps the -35 promoter element and binding of arginine results in a repositioning of the promoter proximal bound ArgP-arg subunits. This effect was mimicked by the generation of a 10bp insertion mutant (ins-10) in the argO operator that renders its activation by ArgP arginine-independent. ArgP-induced DNA bending of the argO operator by approximately 60° was found to be effector independent. An ArgP:DNA binding stoichiometry of 4:1 indicates binding of four ArgP subunits even to DNA constructs that are truncated for one binding subsite (ΔABS). These results provide insight into the molecular mechanisms of ArgP-mediated regulation and a molecular explanation for the unique arginine-dependence of argO activation that distinguishes this particular ArgP target from all others.

Keywords: Amino acid transport; DNA bending; DNA footprinting; IciA; argO; lysP.

MeSH terms

  • Amino Acid Transport Systems, Basic / genetics
  • Arginine / metabolism
  • Bacterial Proteins / genetics*
  • Base Sequence
  • Binding Sites / genetics
  • DNA Footprinting
  • DNA, Bacterial / genetics
  • DNA-Binding Proteins / genetics*
  • Deoxyribonuclease I
  • Escherichia coli / enzymology
  • Escherichia coli / genetics*
  • Escherichia coli Proteins / genetics*
  • Gene Expression Regulation, Bacterial*
  • Lysine / metabolism
  • Mutagenesis, Site-Directed
  • Promoter Regions, Genetic
  • beta-Galactosidase / genetics

Substances

  • Amino Acid Transport Systems, Basic
  • Bacterial Proteins
  • DNA, Bacterial
  • DNA-Binding Proteins
  • Escherichia coli Proteins
  • lysP protein, E coli
  • argP protein, E coli
  • Arginine
  • Deoxyribonuclease I
  • beta-Galactosidase
  • Lysine