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Abstract
The processing or decay of RNA in Escherichia coli involves an array of endonucleases and exonucleases. In addition, the decay of RNA transcripts also involves the post-transcriptional addition of 3 poly(A) tails to mRNAs, rRNAs, and pre-tRNAs by poly(A) polymerase I (PAP I) and polynucleotide phosphorylase (PNPase). The addition of poly(A) tails helps target the RNAs for more rapid degradation. In this work, the processing and/or breakdown along with polyadenylation of the large polycistronic rpsJ mRNA transcript has been examined.Our data suggest that RNase E is the major enzyme involved in the initial processing of the rpsJ transcript and is responsible for the removal of the 3' Rho-independent transcription terminator. The results also suggest that the 5' untranslated region of the rpsJ transcript remains intact in wild-type cells. Additionally, the processing pattern of the rpsJ transcript indicates that other endonucleases generate secondary cleavages. Interestingly, the cell maintains constant ratios of the individual ORF transcripts of the polycistronic rpsJ operon, despite changes in the processing of thefull-length transcript in RNase E mutants. These results suggest a greater flexibility in controlling ribosomal protein transcript levels than previously observed.Furthermore, through our experiments involving the polyadenylation of the rpsJ operon transcript, we show that polyadenylation of mRNAs by PAP I is directly linked to an intact RNase E-based degradosome. The polyadenylation profiles of individual mRNAs encoded within the rpsJ operon (rpsJ and rpsQ) and a control transcript (lpp) changed dramatically both in the location of the tails and in their nucleotide composition when the scaffold region of the RNase E protein was deleted. Specifically, in the absence of degradosome assembly, the majority of the tails for these specific mRNA transcripts were heteropolymeric and were added by PNPase. Our data suggest the existence of a much larger polyadenylation complex and a more direct link between mRNA degradation and polyadenylation than previously envisioned.