Expression of gene 19 of the conjugative plasmid R1 is controlled by RNase III

Specific cleavage of mRNAs by RNase III has been shown to control the expression of several Escherichia coli genes. We show here that the expression of gene 19 of the conjugative resistance plasmid R1 is controlled in its expression by the same endoribonuclease. In vivo studies revealed that a DNA fragment of 150 nucleotides including a perfect 22 nucleotide inverted repeat in the gene 19 coding region is responsible for the low expression of the gene both at the protein and the RNA levels. By using a translational gene 19-lacZ fusion in isogenic RNase III+ and RNase III- strains we could identify RNase III as the key element in the down-regulation of gene 19 expression. The sequencing of in vitro generated and RNase III-digested transcripts confirmed the in vivo studies and revealed the exact positions of the RNase III cleavage sites within the coding part of the gene 19 transcript. The in vitro determined RNase III cleavage of gene 19 mRNA was confirmed by in vivo primer extension analysis. Finally, we could show that an exchange of three nucleotides within the RNase III recognition site abolished RNase III cleavage in vitro.