micF RNA binds to the 5' end of ompF mRNA and to a protein from Escherichia coli

Abstract

MicF RNA regulates the levels of outer membrane protein F (OmpF) in Escherichia coli in response to temperature increase and other stress conditions by decreasing the levels of ompF mRNA (Andersen et al., 1989). A 93-nucleotide micF RNA was synthesized in vitro directly from polymerase chain reaction generated DNA which was designed to contain a functional T7 RNA polymerase promoter upstream of the micF RNA gene and an appropriate restriction site for transcription termination. A transcript (150 nucleotides) containing the ribosomal binding domain of ompF mRNA messenger was synthesized in vitro from the ompF gene cloned into a T7 expression vector. A stable duplex was formed between micF RNA and the 150-nucleotide 5'' transcript of ompF mRNA after incubation at 37.degree. C in a physiological buffer. The melting curve of the duplex formed by micF RNA and 150-nucleotide transcript revealed a Tm of 56.degree. C and a .DELTA.Tm that spans about 20.degree. C; both are consistent with the proposed structure for the micF/ompF duplex. In addition, as determined by competition studies and UV cross-linking/label-transfer analyses, an E. coli protein was found to bind specifically to micF RNA. The protein also bound weakly to the 150-nucleotide ompF transcript. The data are the first to demonstrate the complex between micF RNA and the 5'' end of ompF mRNA and suggest that in vivo of micF ribonucleoprotein (RNP) particle may participate in the destabilization ompF mRNA during thermoregulation of OmpF porin.