sRNA expedites polycistronic mRNA decay in Escherichia coli.

In bacteria, most small RNA (sRNA) elicits RNase E-mediated target mRNA degradation by binding near the translation initiation site at the 5' end of the target mRNA. Spot 42 is an sRNA that binds in the middle of the gal operon near the translation initiation site of galK, the third gene of four, but it is not clear whether this binding causes degradation of gal mRNA. In this study, we measured the decay rate of gal mRNA using Northern blot and found that Spot 42 binding caused degradation of only a specific group of gal mRNA that shares their 3' end with full-length mRNA. The results showed that in the MG1655Δspf strain in which the Spot 42 gene was removed, the half-life of each gal mRNA in the group increased by about 200% compared to the wild type. Since these mRNA species are intermediate mRNA molecules created by the decay process of the full-length gal mRNA, these results suggest that sRNA accelerates the mRNA decaying processes that normally operate, thus revealing an unprecedented role of sRNA in mRNA biology.

Translation Initiation Control of RNase E-Mediated Decay of Polycistronic gal mRNA.

In bacteria, mRNA decay is a major mechanism for regulating gene expression. In Escherichia coli, mRNA decay initiates with endonucleolytic cleavage by RNase E. Translating ribosomes impede RNase E cleavage, thus providing stability to mRNA. In transcripts containing multiple cistrons, the translation of each cistron initiates separately. The effect of internal translation initiations on the decay of polycistronic transcripts remains unknown, which we have investigated here using the four-cistron galETKM transcript. We find that RNase E cleaves a few nucleotides (14-36) upstream of the translation initiation site of each cistron, generating decay intermediates galTKM, galKM, and galM mRNA with fewer but full cistrons. Blocking translation initiation reduced stability, particularly of the mutated cistrons and when they were the 5'-most cistrons. This indicates that, together with translation failure, the location of the cistron is important for its elimination. The instability of the 5'-most cistron did not propagate to the downstream cistrons, possibly due to translation initiation there. Cistron elimination from the 5' end was not always sequential, indicating that RNase E can also directly access a ribosome-free internal cistron. The finding in gal operon of mRNA decay by cistron elimination appears common in E. coli and Salmonella.