A model mRNA, designated DeltaermC mRNA, was used to study the effects of translational signals and ribosome transit on mRNA decay in Bacillus subtilis. Inactivation of the Shine-Dalgarno (SD) sequence or the start codon resulted in a significant decrease in the mRNA half-life, demonstrating the importance of ternary complex formation for mRNA stability. The stability of DeltaermC mRNA was not affected by the insertion of premature stop codons, showing that actual translation was not required for stability. The data are consistent with a model in which ribosome binding and the formation of the ternary complex interfere with the binding of a 5'-end-dependent endoribonuclease, which is required for the initiation of mRNA decay. This model was supported by the finding that increasing the distance from the 5' end to the SD sequence resulted in a decrease in mRNA half-life. Additionally, it was shown that the presence of a 5'-terminal stem-loop structure resulted in a large increase in mRNA stability. An analysis of the context in which the stabilizing structure occurred included the effects of distance from the 5' end, translation of downstream coding sequence, and distance to the SD sequence. These results also underscored the importance of the 5' end to mRNA stability in B. subtilis to which it is hypothesized a 5'-end-dependent endoribonuclease binds. Cleavage by this endoribonuclease may generate accessible 3' ends from which 3'-to-5' exoribonucleases can initiate decay. Previous work has shown that the major 3'-to-5' exoribonuclease in Bacillus subtilis is polynucleotide phosphorylase (PNPase). However, it remains to be determined whether PNPase initiates decay directly from the native 3' end of an mRNA (which is often protected by strong secondary structure that constitutes the transcription terminator), or if decay initiates from a nascent 3' end created by prior endoribonuclease cleavage. To address this question, we analyzed the decay pattern of model mRNAs that contained a stem-loop structure that was able to block PNPase. These data suggested a prior endoribonuclease cleavage upstream of the 3' proximal stem-loop was required to generate an accessible 3' end from which PNPase could initiate degradation.
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