Overexpression and DNA-binding properties of the mer-encoded regulatory protein from plasmid NR1 (Tn21).

A Heltzel,A O Summers,P A Totis,D Gambill,W J Jackson

doi:10.1128/jb.169.7.3379-3384.1987

Abstract

In plasmid NR1 the expression of genes involved in mercury resistance (Tn21) is regulated by the trans-acting product of the merR gene. An in vivo T7 RNA polymerase-promoter overexpression system was used to detect a protein of approximately 16,000 daltons encoded by the merR reading frame. Overexpressed MerR constituted about 5% of labeled proteins. An in vitro MerR-mer-op (mer-op is the mer operator and promoter region) gel electrophoresis binding assay established that the binding site for MerR was located between the putative -35 and -10 sequences of the promoter for the mer structural genes. A nonsense mutation in the carboxyl half of MerR resulted in the loss of biological function and the loss of in vitro mer-op binding properties.

Highlights

In plasmid NR1 the expression of genes involved in mercury resistance (Tn2l) is regulated by the trans-acting product of the merR gene
An in vitro MerR-mer-op gel electrophoresis binding assay established that the binding site for MerR was located between the putative -35 and -10 sequences of the promoter for the mer structural genes
The two smaller (6,457and 13,139-dalton) polypeptides predicted from the open reading frames (ORFs) are oriented in the same direction as the mer structural genes

Summary

Hinc E

Site before termination at a TGA codon in frame with ORF 3. The 665-bp ScaI-NcoI fragment contains the entire merR gene, the putative transcriptional regulatory site of the operon, and the first 30 amino acid residues of merT [1]. Cells were incubated for an additional 15 min at 42°C, and the temperature was shifted to 30°C for 20 min before the cells were pulse-labeled for 5 min with 15 ,uCi of [35S]cysteine (Amersham Corp., Chicago, Ill.) After they were harvested, the cells were suspended in 50 ,ul of Na2PO4 buffer (pH 7.2), and phenylmethylsulfonyl fluoride was added to a final concentration of 2.5 mM. Densitometric analysis of gels prepared by using several dilutions of labeled protein showed that the expression level of the labeled 16,000-dalton merR protein was about 10% of ,-lactamase (data not shown).

Hinf I

IA BCIEF

LITERATURE CITED