George A. Mackie

The RNA degradosome is a multiprotein complex required for the degradation of highly structured RNAs. We have developed a method for reconstituting a minimal degradosome from purified proteins. Our results demonstrate that a degradosome-like complex containing RNase E, PNPase, and RhlB can form spontaneously in vitro in the absence of all other cellular components. Moreover, ATP-dependent degradation of themalEF REP RNA by the reconstituted, minimal degradosome is indistinguishable from that of degradosomes isolated from whole cells. The Rne protein serves as an essential scaffold in the reconstitution process; however, RNase E activity is not required. Rather, Rne coordinates the activation of RhlB dependent on a 3′ single-stranded extension on RNA substrates. A model for degradosome-mediated degradation of structured RNA is presented with its implications for mRNA decay in Escherichia coli.

The selective degradation of messenger RNAs enables cells to regulate the levels of particular mRNAs in response to changes in the environment. Ribonuclease (RNase) E (ref. 1), a single-strand-specific endonuclease^2,3,4 that is found in a multi-enzyme complex known as the ‘degradosome’^5,6,7, initiates the degradation of many mRNAs in Escherichia coli³,⁸,⁹. Its relative lack of sequence specificity and the presence of many potential cleavage sites in mRNA substrates²,³ cannot explain why mRNA decay frequently proceeds in a net 5′-to-3′ direction^9,10,11. I have prepared covalently closed circular derivatives of natural substrates, the rpsT mRNA encoding ribosomal protein S20 (ref. 2) and the 9S precursor to 5S ribosomal RNA¹,¹², and find that these derivatives are considerably more resistant to cleavage in vitro by RNase E than are linear molecules. Moreover, antisense oligo-deoxynucleotides complementary to the 5′ end of linear substrates significantly reduce the latter's susceptibility to attack by RNase E. Finally, natural substrates with terminal 5′-triphosphate groups are poorly cleaved by RNase E in vitro, whereas 5′ monophosphorylated substrates are strongly preferred (compare with ref. 13). These results show that RNase E has inherent vectorial properties, with its activity depending on the 5′ end of its substrates; this can account for the direction of mRNA decay in E. coli, the phenomenon of ‘all or none’ mRNA decay, and the stabilization provided by 5′ stem–loop structures^14,15,16,17.