1. Department of Biochemsitry & Molecular Biology, University of British Columbia, 2146 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada

Correspondence to: George A. Mackie¹ Correspondence and requests for materials should be addressed to the author
(e-mail: Email: gamackie@unixg.ubc.ca).

Abstract

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}.