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Abstract In Escherichia coli, RNase E is the key enzyme for RNA processing and mRNA degradation. Despite the conserved function across bacteria, the domain composition of RNase E varies significantly among species, possibly affecting the enzyme’s subcellular localization, mobility, and function. In this work, we used super-resolution microscopy to find that 93% of RNase E is localized to the membrane in E. coli and exhibits slow diffusion comparable to polysomes diffusing in the cytoplasm. By replacing the native amphipathic membrane targeting sequence (MTS) with a transmembrane motif, we discovered that the MTS results in slower diffusion and stronger membrane binding than a transmembrane motif. Additionally, the evolutionarily divergent C-terminal domain (CTD) was shown to grant slow diffusion of RNase E but to weaken its membrane binding. By analyzing how membrane localization and diffusion of RNase E affect mRNA degradation rates in vivo, we provide new insights into RNase E’s role in the spatiotemporal organization of RNA processes in bacterial cells. 

Abstract In eukaryotic cells, transcription, translation, and mRNA degradation occur in distinct subcellular regions. How these mRNA processes are organized in bacteria, without employing membrane-bound compartments, remains unclear. Here, we present generalizable principles underlying coordination between these processes in bacteria. InEscherichia coli, we found that co-transcriptional degradation is rare for mRNAs except for those encoding inner membrane proteins, due to membrane localization of the main ribonuclease, RNase E. We further found, by varying ribosome binding sequences, that translation affects mRNA stability not because ribosomes protect mRNA from degradation, but because low translation leads to premature transcription termination in the absence of transcription-translation coupling. Extending our analyses toBacillus subtilisandCaulobacter crescentus, we established subcellular localization of RNase E (or its homolog) and premature transcription termination in the absence of transcription-translation coupling as key determinants that explain differences in transcriptional and translational coupling to mRNA degradation across genes and species.