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Reconstitution of the decay of the rpsT mRNA, encoding ribosomal protein S20, with purified enzymes

University of British Columbia

Metabolic instability, a hallmark property of all mRNAs, can impart a number of important consequences on the regulation of gene expression. To further study the process of mRNA degradation in Escherichia coli, we have developed an in vitro system to determine the requirements for the complete decay of the rpsTmKNA, encoding ribosomal protein S20. The data show that both purified RNase II and polynucleotide phosphorylase can catalyze the degradation of the 5'-twothirds of the rpsT mRNA and that prior oligoadenylation of the 3'-termini of truncated rpsT mRNA substrates can stimulate significantly the initiation of degradation by the 3'-exonucleases. The intact rpsT mRNA, however, is insensitive to attack by RNase II or polynucleotide phosphorylase. Furthermore, the single addition of a poly (A) tail to the 3'-end of the rpsT mRNA cannot overcome its resistance to either 3'-exonuclease in vitro. Although previous work has implicated the product of the pcnB gene in the decay of a number of RNAs from Escherichia coli, poly(A) polymerase I does not promote the initiation of the decay of the rpsT mRNA in vivo. It does, however, facilitate the degradation of highly folded degradative intermediates by polynucleotide phosphorylase. As expected, purified degradosomes 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 polynucleotide phosphorylase 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, ATPyS, support the poly (A) polymerase I and polynucleotide phosphorylase-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 poly(A) polymerase I, polynucleotide phosphorylase and ATP alone. Complete degradation of a fragment of the malE-malF mRNA in vitro exhibits additional requirements. Degradation of this RNA is dependent on the degradosome, ATP and poly(A) polymerase I. Unlike the situation for the rpsT mRNA, the non-hydrolyzable analog, ATPyS, cannot substitute for ATP suggesting that ATP hydrolysis is required for decay of structured portions of the malE-malF mRNA. 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. The implications of these findings are discussed in a number of models of mRNA decay.

Thesis/Dissertation

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2009-06-19

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http://hdl.handle.net/2429/9477

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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