Reconstitution of the degradation of the mRNA for ribosomal protein S20 with purified enzymes

• Published in: Journal of molecular biology 1998-06, Vol.279 (5), p.1061-1074
• Main Authors: Coburn, Glen A, Mackie, George A
• Format: Article
• Language: English
• Subjects: Blotting, Northern; degradosome; Endoribonucleases - metabolism; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - metabolism; mRNA decay; Multienzyme Complexes - metabolism; Nucleic Acid Conformation; PNPase purification; polyadenylation; Polynucleotide Adenylyltransferase - metabolism; Polyribonucleotide Nucleotidyltransferase - metabolism; Ribosomal Proteins - genetics; Ribosomal Proteins - metabolism; RNA Helicases; RNA, Bacterial - chemistry; RNA, Bacterial - metabolism; RNA, Messenger - metabolism
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.1998.1842

Previous work has implicated poly(A) polymerase I (PAP I), encoded by the pcnB gene, in the decay of a number of RNAs from Escherichia coli. We show here that PAP I does not promote the initiation of decay of the rpsT mRNA encoding ribosomal protein S20 in vivo; however, it does facilitate the degradation of highly folded degradative intermediates by polynucleotide phosphorylase. As expected, purified degradosomes, a multi-protein complex containing, among others, RNase E, PNPase, and RhlB, generate an authentic 147-residue RNase E cleavage product from the rpsT mRNA in vitro. However, degradosomes are unable to degrade the 147-residue fragment in the presence of ATP even when it is oligoadenylated. Rather, both continuous cycles of polyadenylation and PNPase activity are necessary and sufficient for the complete decay of the 147-residue fragment in a process which can be antagonized by the action of RNase II. Moreover, both ATP and a non-hydrolyzable analog, ATPγS, support the PAP I and PNPase-dependent degradation of the 147-residue intermediate implying that ATPase activity, such as that which may reside in RhlB, a putative RNA helicase, is not necessarily required. Alternatively, the rpsT mRNA can be degraded in vitro by a second 3′-decay pathway which is dependent on PAP I, PNPase and ATP alone. Our results demonstrate that a hierarchy of RNA secondary structures controls access to exonucleolytic attack on 3′ termini. Moreover, decay of a model mRNA can be reconstituted in vitro by a small number of purified components in a process which is more dynamic and ATP-dependent than previously imagined.