| The Selective RNA Processing and Stabilization mechanism(SRPS)is an important posttranscriptional regulation mechanism of microbial protein complex subunits stoichiometry.It resolves the contradiction between simultaneous transcription of genes in the operon and the need for differential expression of each gene.How the coordinated actions of SRPS-related enzymes affect stoichiometric regulation has not been investigated.Here,we report the first genome-wide targetomes of these enzymes in E.coli at single-nucleotide resolution,unveil the interplay between the pyrophosphohydrolase,endoRNase and exoRNases,depict the molecular regulation model of SRPS in E.coli.The targetomes were shown to be enriched in operons,especially those exhibiting a highly skewed transcript abundance landscape.We demonstrate that RNase E preferentially cleaves before two uridines and that the newly generated 3’ ends are devoured by PNPase,nibbled by RNase Ⅱ,and occasionally trimmed by RNase R.A strictly linear relationship was observed between the RppH processing ratio and the scores assigned to the first three nucleotides on the primary transcript.Moreover,stem-loops associated with PNPase targetomes exhibited a folding free energy that was negatively correlated with the termination ratio of PNPase at the 3’ terminus.More than one-tenth of the RNase E processing sites in 5’ untranslated regions formed different stem-loops that affected ribosome binding and translation efficiency.We then depicted the stoichiometric regulatory model of SRPS in E.coli.The effectiveness of several SRPS elements was validated by using a dual-fluorescence reporter system.Finally,we constructed the first prokaryotic SRPS site database.Our results highlight a multilayer and quantitative regulatory method for optimizing the stoichiometric expression of genes in bacteria and promote the application of SRPS in synthetic biology. |
