Rho-dependent termination and RNase E-mediated cleavage: Dual pathways for RNA 3' end processing in polycistronic mRNA

Abstract

"Pre-full-length" transcripts are produced at the end of the polycistronic galactose (gal) operon, 5galE-galT-galK-galM3, via Rho-dependent (RDT) and -independent transcription termination (RIT). The full-length galETKM mRNAs 3 end is acquired by exo-nucleolytic processing of the 3-OH ends of the pre-full-length transcripts. However, the gal operon produces an mRNA named galE whose 3 end forms at [~]120 nucleotides from the galE stop codon, thus in the following gene, galT, establishing polarity in gene expression. In this study, we investigated the molecular processes that generate the 3 end of galE mRNA. We discovered that the 3 ends of pre-galE mRNA are produced in the middle of the galT gene as a result of the combination of two separate molecular processes - one previously reported as RDT and the other as unreported RNase E-mediated transcript cleavage. The 3 ends of the pre-galE mRNA are exo-nucleolytically processed to the current 3 end of the galE mRNA. A hairpin structure of 8 base-pair stems and 4 nucleotide-loop formed 5-10 nucleotides upstream of the 3 ends of the galE mRNA blocks the exoribonuclease digestion and renders stability. These findings showed that RNase E produces RNA 3end establishing polarity in gene expression, in contrast to the general role of mRNA degradation. Significance statementHere, we show the findings of two molecular mechanisms that generate the pre-galE mRNA 3ends in the gal operon: Rho-dependent termination (RDT) and RNase E-mediated cleavage. These 3 ends are subsequently processed to produce stable galE mRNA with a hairpin structure that prevents exoribonuclease degradation. This mechanism establishes gene expression polarity by generating the 3 end of galE mRNA within the galT gene, contrasting with the usual mRNA degradation role of RNase E. The study reveals a unique role of RNase E in mRNA processing and stability.