Effect of Integration Host Factor on RNA II Synthesis in Replication of Plasmid Containingorip15A

Abstract

The synthesis rates of the replication control RNAs of plasmidorip15A, RNA I, an inhibitor of replication, and RNA II, the primer, have been determined usinglacZfusion plasmids, hybridization assay, and reverse transcription polymerase chain reaction (RT-PCR) inEscherichia coliintegration host factor-positive (IHF+) and -negative (IHF−) strains containing pACYC184 plasmid (orip15A). In the absence of IHF (E. coliIHF−), expression of thelacZgene from thePRNAIIpromoter increased by a factor of 4 compared with theE. coliwild type (IHF+). Also, the increase in expression was more pronounced when the IHF protein was mutated in theihfBgene than in theihfAgene. For thePRNAIIpromoter oforipMB1 (pBR322), no significant differences were found in expression of thelacZgene in heE. colistrains examined. The level of β-galactosidase expression from thePRNAIpromoter oforip15A shows that the absence of functional IHF in the transformed strains has no effect on expression of thelacZgene. The synthesis RNA II:RNA I ratio obtained in hybridization assays was 2.4 forE. coliIHF+and 4.4 forE. coliIHF−. Densitometric analysis of RT-PCR products indicates that the relative levels of RNA I inE. coliIHF+and IHF−, are equal, but the relative level of RNA II inE. coliIHF−is about four times higher than inE. coliIHF+. These results indicatethat the IHF protein inhibits transcription from thePRNAIIpromoter oforip15A plasmid.