Regulation of the ompC gene of Escherichia coli. Involvement of three tandem promoters.

K Ikenaka,G Ramakrishnan,K Tsung,M Inouye

doi:10.1016/s0021-9258(18)67656-2

Abstract

ompC expression in Escherichia coli K-12 is known to be regulated by the ompB locus, comprising the ompR and envZ genes, and the OmpR protein is believed to act as a positive transcriptional factor. We examined the transcriptional capability of the ompC gene in vitro and found that RNA polymerase could transcribe ompC without a requirement for other transcriptional factors. Furthermore, transcripts from three tandem promoters in ompC were identified in vitro. We employed oligonucleotide-directed site-specific mutagenesis to dissect the promoter region of the gene and assayed the promoters separately for transcriptional ability using fusions to the lacZ gene. The levels of beta-galactosidase indicate that ompC expression in vivo is dependent on the function of at least one of the upstream promoters. The function of OmpR appears to be the enhancement of a basal level of ompC expression. From the results of our experiments, the site of action of OmpR was deduced to be in the vicinity of the upstream promoters of ompC.

Highlights

From the Department of Biochemistry, State University of New York at Stony Brook, Stony Brook, New York 11794
OmpC expression inEscherichia coliK- 12 is known As a direct test of this hypothesis, we have undertaken in toberegulatedbythe ompB locus,comprisingthe vitro transcriptional studiesand found that RNA polymerase ompR and envZ genes, and the OmpR protein is be- can transcribe ompC without the aid of positive factors
We we found transcription to occur from multiple examined the transcriptional capability of the ompC tandem promoters, which proved to be consistent with the sgcgtstvrhccieeifarrtrnnirniieceeppoesmtt.caitiinraW noouinnndbtvedaaaeiafegllteoasrmamesmconbatpppeoiyallCrsreionoitsdysdwFym.etufitudhotoorhsteuotieohnlndpruiegdisrgstriotsofnmhmeunaacuosottcirrootleetemnet,hRqrrospesuatNCtinisoArpdeseeprwctm-porhaedmioreeriplanlreytoatetfsmtecrciedlZotfyeroeemrgrnfdaroestesnriioegtfeteiticehr.o-odsaeTnuprnhlie-done-tfrathnervbsomeesfiae-dsoiqsstuutauhellateitroslsnlsle.ycisvsneeheftpooelofartrwharttacarhttatteheinoasritnesthcsgreauoiisppobmeptnjpieep.oCcarWntorastmieols ohtaaeotaccxevttrpbiiesvverieauattstysnihsoedeeidndnhusbvaapiytvitsetetOr-raodetmieaalrosmpenwtRcedtd,teicaadntonnhndmvdeseimuvtmitottha.uigenptOsiedrvionutiee---r levels of @-galactosidase indicate othmaptC expression in vivo is dependent on the function ofat least one of the upstream promoters

Summary

Introduction

The 350-bp XbaI fragment carried in pKL007 was cloned into vector pJDC406 to generate plasmid pKI0641which wassubsequently employed in site-directed mutagenesis. In order to test in vivo activities of the dissected ompC promoter, each of the mutant plasmids pKI0643, pKI0644, and pKI0645 was digested with XbaI and the particular enzyme whose site was introduced by mutagenesis, and thefragments carrying the start of transcriptional of the ompC gene were cloned into pKM007 to produce plasmids pKI0743, pKI0744, and pKI0745, respectively (Fig. 1C).

Results

Conclusion