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Mackie, GA (1998) Ribonuclease E is a 5'-end-dependent endonuclease. Nature 395:720-3
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, a single-strand-specific endonuclease that is found in a multi-enzyme complex known as the 'degradosome', initiates the degradation of many mRNAs in Escherichia coli. Its relative lack of sequence specificity and the presence of many potential cleavage sites in mRNA substrates cannot explain why mRNA decay frequently proceeds in a net 5'-to-3' direction. I have prepared covalently closed circular derivatives of natural substrates, the rpsT mRNA encoding ribosomal protein S20 and the 9S precursor to 5S ribosomal RNA, 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. 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.
Binding Sites; Endoribonucleases/antagonists & inhibitors; Endoribonucleases/chemistry; Endoribonucleases/metabolism; Escherichia coli/enzymology; Nucleic Acid Heteroduplexes/metabolism; Oligodeoxyribonucleotides, Antisense/pharmacology; RNA/metabolism; RNA, Messenger/metabolism; Ribosomal Proteins/genetics; Substrate Specificity
|Gene product||Qualifier||GO Term||Evidence Code||with/from||Aspect||Extension||Notes||Status|
|GO:0004519: endonuclease activity||
Two forms of RNA were used, linear and circular. The circular RNA did not have a 5' end and thus could not be digested by RNase E as shown in Figure 2a. The circular RNA was then digested by RNase H and thus linearized as shown in Figure 2b. The now linearized RNA was then digested by RNase E which since the RNA was linear could be digested by RNase E as shown in Figure 2d and Figure 2e. The bands shown in Figures 2d,e are consistent with the digestion sites of both RNase H and RNase E.
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