UBC Theses and Dissertations
A novel mechanism for RNase E-initiated decay of 5’-protected mRNA in Escherichia coli Baker, Kristian Eileen
In Escherichia coli, 5'-terminal RNA stem-loop structures are major impediments to the rate-determining initiation of mRNA degradation catalyzed by the endonuclease RNase E. Protected RNAs do, nonetheless, decay at appreciable rates, and factors which enforce the initial barrier to decay or modulate an alternative decay pathway are unknown. A prosthetic RNA hairpin at the extreme 5'-terminus of a model mRNA substrate, r/w7mRNA, stabilized this RNA 6-fold against a decay-initiating RNase E cleavage. Altering the efficiency of translation or introducing premature-termination codons demonstrated that stem-loop-mediated stabilization of rpsT mRNA depended on efficient translational initiation and ribosome passage through the ratedetermining RNase E cleavage site within the coding sequence. Introduction of sequences encompassing characterized RNase E cleavage sites, referred to here as 'portable' RNase E cleavage sites, from either the me mRNA leader or 9S rRNA into rpsT mRNA significantly reduced the stabilizing effects of the terminal stem-loop. Destabilization by the 'portable' cleavage sequences required active RNase E, and cleavage-site mapping demonstrated endonucleolytic cleavage within the inserted sequences. Furthermore, the efficiency of destabilization by 'portable' RNase E cleavage sites was dependent on both the primary sequence of the insert as well as its position within the 5' UTR or ORF of the stem-loop-protected rpsT mRNA. This work has led to the proposal of a model for RNase E-mediated degradation of 5'- protected mRNA involving bypass of the preferred interaction with the 5' terminus and 'internal' recognition of the mRNA substrate. Moreover, the efficiency of'internal' entry appears to be determined by the intrinsic susceptibility of the rate-limiting cleavage site alone. Finally, the involvement of the arginine-rich RNA binding domain of RNase E in the internal entry mechanism of RNA degradation is discussed.
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