Two targets in pCF10 DNA for PrgX binding: their role in production of Qa and prgX mRNA and in regulation of pheromone-inducible conjugation

J Mol Biol. 2002 Feb 1;315(5):995-1007. doi: 10.1006/jmbi.2001.5294.

Abstract

PrgX is the primary cytoplasmic protein involved in negative control of pheromone-inducible conjugation functions of the Enterococcus faecalis plasmid pCF10. PrgX is believed to act in concert with an antisense RNA called Qa to inhibit readthrough of transcription from the prgQ promoter into the pCF10 genes mediating conjugation functions; PrgX also positively regulates its own expression, as well as that of Qa. We found two DNA target sites for PrgX binding in the intergenic region between the prgX and prgQ genes of pCF10. The primary binding site near prgX includes an 11 bp palindromic sequence and showed relatively high affinity for His-tagged PrgX (His-PrgX). The secondary binding site is between the -35 and -10 regions of the prgQ promoter, and contains only a half of the palindromic sequence; this binding site showed weaker affinity. A region of pCF10 including the prgQ promoter and the secondary binding site reduced Qa RNA levels greatly and this reduction was overcome by the presence of the primary binding site and PrgX. In constructs where the binding sites were mutated individually or in combination, the intracellular levels of PrgX protein and Qa RNA were reduced significantly. On the basis of these results, we propose that both DNA binding sites are required for the autoregulation of PrgX expression and for positive regulation of Qa RNA.

Publication types

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

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Base Sequence
  • Binding Sites
  • Conjugation, Genetic / drug effects*
  • DNA Footprinting
  • DNA, Intergenic / genetics
  • DNA, Intergenic / metabolism
  • DNA-Binding Proteins / metabolism*
  • Electrophoretic Mobility Shift Assay
  • Enterococcus faecalis / drug effects
  • Enterococcus faecalis / genetics*
  • Gene Expression Regulation, Bacterial / drug effects
  • Genes, Bacterial / genetics*
  • Molecular Sequence Data
  • Mutation / genetics
  • Pheromones / pharmacology*
  • Plasmids / genetics
  • Plasmids / metabolism*
  • Promoter Regions, Genetic / genetics
  • Protein Binding
  • Protein Sorting Signals / genetics
  • RNA, Antisense / biosynthesis
  • RNA, Antisense / genetics
  • RNA, Bacterial / biosynthesis*
  • RNA, Bacterial / genetics
  • Response Elements / genetics
  • Thermodynamics

Substances

  • Bacterial Proteins
  • DNA, Intergenic
  • DNA-Binding Proteins
  • Pheromones
  • Protein Sorting Signals
  • RNA, Antisense
  • RNA, Bacterial
  • iCF10 peptide precursor, Plasmid pCF10