In vitro analysis of mRNA processing by RNase E in the pap operon of Escherichia coli

Differential gene expression from operons encoding fimbrial adhesins in Escherichia coli involves processing and differential decay of polycistronic transcripts. Previous analyses of mRNA processing in vivo using ribonuclease mutants of E. coli have given different results with the different fimbrial gene systems tested. For the pap operon from uropathogenic E. coli, the results suggested that the mRNA processing is dependent on ribonuclease E (RNase E), whereas in other fimbrial operons with similar genetic organisation, the processing was concluded to be RNase E independent. We have developed an in vitro system allowing us to assess the cleavage of pap mRNA, to study the mRNA processing of a fimbrial operon in more detail, and to define the enzymatic activity and target. The results of this study establish that RNase E does indeed cleave the papBA intercistronic transcript. Analysis of the cleavage products reveals that in vitro RNase E can cleave the mRNA at other positions in addition to the site preferentially cleaved in vivo. The specificity of the cleavage pattern was assessed using transcripts derived from mutants with base substitutions near, or within, the major in vivo cleavage site. Such mutants have alternative cleavage sites. A common feature of the different cleavage sites is a high A/U nucleotide content, similar to other known RNase E cleavage sites. Features of the secondary structure of the papBA intercistronic mRNA were investigated using single-strand-specific and double-strand-specific nucleases. The secondary structure model derived from stability calculations and our results from the nuclease-probing experiments indicate that the positions subject to RNase E cleavage are mainly single stranded and flanked by more stable stem-loop structures. The results are consistent with the notion that an mRNA conformation exposing A/U-rich, non-paired regions constitutes the target, i.e. a flexible determinant, for processing by RNase E in the pap transcript. The findings are discussed in relation to the existence of a potential recognition site for RNase E and the analysis of RNase E cleavages in other RNA molecules.