IN-VITRO ANALYSIS OF MESSENGER-RNA PROCESSING BY RNASE-E IN THE PAP OPERON OF ESCHERICHIA-COLI

Differential gene expression from operons encoding fimbrial adhesins i n Escherichia coil involves processing and differential decay of polyc istronic transcripts, Previous analyses of mRNA processing in vivo usi ng ribonuclease mutants of E. coil have given different results with t he different fimbrial gene systems tested, For the pap operon from uro pathogenic E. coil, the results suggested that the mRNA processing is dependent on ribonuclease E (RNase E), whereas in other fimbrial opero ns 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 activit y and target, The results of this study establish that RNase E does in deed cleave the papBA intercistronic transcript. Analysis of the cleav age products reveals that in vitro RNase E can cleave the mRNA at othe r positions in addition to the site preferentially cleaved in vivo, Th e specificity of the cleavage pattern was assessed using transcripts d erived 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 nucleot ide content, similar to other known RNase E cleavage sites. Features o f the secondary structure of the papBA intercistronic mRNA were invest igated using single-strand-specific and double-strand-specific nucleas es, The secondary structure model derived from stability calculations and our results from the nuclease-probing experiments indicate that th e positions subject to RNase E cleavage are mainly single stranded and flanked by more stable stem-loop structures. The results are consiste nt with the notion that an mRNA conformation exposing A/U-rich, non-pa ired regions constitutes the target, i.e. a flexible determinant, for processing by RNase E in the pap transcript. The findings are discusse d in relation to the existence of a potential recognition site for RNa se E and the analysis of RNase E cleavages in other RNA molecules.