Purification and characterization of a novel organometallic receptor protein regulating the expression of the broad spectrum mercury-resistant operon of plasmid pDU1358

Hongri Yu, Debabrata Mukhopadhyay, Tapan K. Misra

Research output: Contribution to journalArticle

15 Scopus citations

Abstract

The narrow spectrum mercury-resistant (mer) operons of transposons Tn21 and Tn501 are inducible by inorganic mercury salts. The major regulatory gene merR is transcribed divergently from the other mer genes, which are cotranscribed. The MerR protein represses its own expression, as well as the expression of the other mer genes in the absence of the inducers. The synthesis of the polycistronic mer message is stimulated by MerR in the presence of the inducers. The MerR(BS) protein encoded by the broad spectrum mer operon of plasmid pDU1358 was characterized as a novel organomercurial receptor, distinguishing it from the narrow spectrum MerR(NS) proteins, described above. Several organomercurial compounds directly effected cellular activation of the mer operon transcription via the receptor protein MerR(BS), but not by MerR(NS). The merR gene from pDU1358 was cloned under the tac promoter, and the overexpressed MerR(BS) protein was soluble in buffer solutions containing 0.5 M NaCl at pH 7.5, but precipitated when NaCl concentration was reduced to 0.1 M (MerR(BS) concentrations at or above 0.1 mg/ml). MerR(BS) was purified to near homogeneity by selective precipitation and solubilization by varying the salt concentration in buffer solutions, followed by Sephadex G-75 column chromatography. Both MerR(BS) and Tn21- encoded MerR(NS) bound with DNA fragments containing the pDU 1358 mer operator sequence with comparable affinities. In vitro run-off transcription studies revealed that MerR(BS) activated mer operon expression in the presence of Hg2+ or phenylmercuric acetate. Phenylmercuric acetate did not induce mer operon expression when the MerR(NS) was used in the assay.

Original languageEnglish (US)
Pages (from-to)15697-15702
Number of pages6
JournalJournal of Biological Chemistry
Volume269
Issue number22
StatePublished - Jun 3 1994

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Purification and characterization of a novel organometallic receptor protein regulating the expression of the broad spectrum mercury-resistant operon of plasmid pDU1358'. Together they form a unique fingerprint.

  • Cite this