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Bacterial mRNAs have short life cycles, in which transcription is rapidly followed by translation and degradation within seconds to minutes. The resulting diversity of mRNA molecules across different life-cycle stages impacts their functionality but has remained unresolved. Here we quantitatively map the 3’ status of cellular RNAs in Escherichia coli during steady-state growth and report a large fraction of molecules (median>60%) that are fragments of canonical full-length mRNAs. The majority of RNA fragments are decay intermediates, whereas nascent RNAs contribute to a smaller fraction. Despite the prevalence of decay intermediates in total cellular RNA, these intermediates are underrepresented in the pool of ribosome-associated transcripts and can thus distort quantifications and differential expression analyses for the abundance of full-length, functional mRNAs. The large heterogeneity within mRNA molecules in vivo highlights the importance in discerning functional transcripts and provides a lens for studying the dynamic life cycle of mRNAs.

 

Bacterial protein synthesis rates have evolved to maintain preferred stoichiometries at striking precision, from the components of protein complexes to constituents of entire pathways. Setting relative protein production rates to be well within a factor of two requires concerted tuning of transcription, RNA turnover, and translation, allowing many potential regulatory strategies to achieve the preferred output. The last decade has seen a greatly expanded capacity for precise interrogation of each step of the central dogma genome-wide. Here, we summarize how these technologies have shaped the current understanding of diverse bacterial regulatory architectures underpinning stoichiometric protein synthesis. We focus on the emerging expanded view of bacterial operons, which encode diverse primary and secondary mRNA structures for tuning protein stoichiometry. Emphasis is placed on how quantitative tuning is achieved. We discuss the challenges and open questions in the application of quantitative, genome-wide methodologies to the problem of precise protein production. 

Enzymatic pathways have evolved uniquely preferred protein expression stoichiometry in living cells, but our ability to predict the optimal abundances from basic properties remains underdeveloped. Here, we report a biophysical, first-principles model of growth optimization for core mRNA translation, a multi-enzyme system that involves proteins with a broadly conserved stoichiometry spanning two orders of magnitude. We show that predictions from maximization of ribosome usage in a parsimonious flux model constrained by proteome allocation agree with the conserved ratios of translation factors. The analytical solutions, without free parameters, provide an interpretable framework for the observed hierarchy of expression levels based on simple biophysical properties, such as diffusion constants and protein sizes. Our results provide an intuitive and quantitative understanding for the construction of a central process of life, as well as a path toward rational design of pathway-specific enzyme expression stoichiometry. 

Sigma factors are an important class of bacterial transcription factors that lend specificity to RNA polymerases by binding to distinct promoter elements for genes in their regulons. Here we show that activation of the general stress sigma factor, σ B , in Bacillus subtilis paradoxically leads to dramatic induction of translation for a subset of its regulon genes. These genes are translationally repressed when transcribed by the housekeeping sigma factor, σ A , owing to extended RNA secondary structures as determined in vivo using DMS-MaPseq. Transcription from σ B -dependent promoters excludes the secondary structures and activates translation, leading to dual induction. Translation efficiencies between σ B - and σ A -dependent RNA isoforms can vary by up to 100-fold, which in multiple cases exceeds the magnitude of transcriptional induction. These results highlight the role of long-range RNA folding in modulating translation and demonstrate that a transcription factor can regulate protein synthesis beyond its effects on transcript levels.