Identifying and characterizing substrates of the RNase E/G family of enzymes.

The study of RNA decay and processing in Escherichia coli has revealed a central role for RNase E, an endonuclease that is essential for cell viability. This enzyme is required for the normal rapid decay of many transcripts and is involved in the processing of precursors of 16S and 5S ribosomal RNA, transfer RNA, the transfer-messenger RNA, and the RNA component of RNase P. Although there is reasonable knowledge of the repertoire of transcripts cleaved by RNase E in E. coli, a detailed understanding of the molecular recognition events that control the cleavage of RNA by this key enzyme is only starting to emerge. Here we describe methods for identifying sites of endonucleolytic cleavage and determining whether they depend on functional RNase E. This is illustrated with the pyrG eno bicistronic transcript, which is cleaved in the intergenic region primarily by an RNase E-dependent activity and not as previously thought by RNase III. We also describe the use of oligoribonucleotide and in vitro-transcribed substrates to investigate cis-acting factors such as 5'-monophosphorylation, which can significantly enhance the rate of cleavage but is insufficient to ensure processivity. Most of the approaches that we describe can be applied to the study of homologs of E. coli RNase E, which have been found in approximately half of the eubacteria that have been sequenced.