Coexpression of proteins in response to pathway-inducing signals is the founding paradigm of gene regulation. However, it remains unexplored whether the relative abundance of co-regulated proteins requires precise tuning. Here, we present large-scale analyses of protein stoichiometry and corresponding regulatory strategies for 21 pathways and 67-224 operons in divergent bacteria separated by 0.6-2 billion years. Using end-enriched RNA-sequencing (Rend-seq) with single-nucleotide resolution, we found that many bacterial gene clusters encoding conserved pathways have undergone massive divergence in transcript abundance and architectures via remodeling of internal promoters and terminators. Remarkably, these evolutionary changes are compensated post-transcriptionally to maintain preferred stoichiometry of protein synthesis rates. Even more strikingly, in eukaryotic budding yeast, functionally analogous proteins that arose independently from bacterial counterparts also evolved to convergent in-pathway expression. The broad requirement for exact protein stoichiometries despite regulatory divergence provides an unexpected principle for building biological pathways both in nature and for synthetic activities.

Accurate measurements of cellular protein concentrations are invaluable to quantitative studies of gene expression and physiology in living cells. Here, we developed a versatile mass spectrometric workflow based on data-independent acquisition proteomics (DIA/SWATH) together with a novel protein inference algorithm (xTop). We used this workflow to accurately quantify absolute protein abundances in Escherichia coli for > 2,000 proteins over > 60 growth conditions, including nutrient limitations, non-metabolic stresses, and non-planktonic states. The resulting high-quality dataset of protein mass fractions allowed us to characterize proteome responses from a coarse (groups of related proteins) to a fine (individual) protein level. Hereby, a plethora of novel biological findings could be elucidated, including the generic upregulation of low-abundant proteins under various metabolic limitations, the non-specificity of catabolic enzymes upregulated under carbon limitation, the lack of largescale proteome reallocation under stress compared to nutrient limitations, as well as surprising strain-dependent effects important for biofilm formation. These results present valuable resources for the systems biology community and can be used for future multi-omics studies of gene regulation and metabolic control in E. coli.

Coupled transcription and translation is considered a defining feature of bacterial gene expression 1 , 2 . The pioneering ribosome can both physically associate and kinetically coordinate with the RNA polymerase (RNAP) 3 - 11 , forming a signal-integration hub for co-transcriptional regulation that includes translation-based attenuation 12 , 13 and RNA quality control 2 . However, whether transcription-translation coupling – together with its broad functional consequences – is indeed a fundamental characteristic outside the well-studied Escherichia coli remains unresolved. Here we show that RNAPs outpace pioneering ribosomes in the Gram-positive model bacterium Bacillus subtilis , and that this ‘runaway transcription’ creates alternative rules for both global RNA surveillance and translational control of nascent RNA. In particular, uncoupled RNAPs in B. subtilis explain a diminished role of Rho-dependent transcription termination, as well as the prevalence of mRNA leaders that utilize riboswitches and RNA-binding proteins. More broadly, we identified widespread genomic signatures of runaway transcription in distinct phyla across the bacterial domain of life. Our results demonstrate that coupled RNAP-ribosome movement is not a general hallmark of bacteria. Instead, translation-coupled transcription and runaway transcription constitute two principal modes of gene expression that determine genome-specific regulatory mechanisms in prokaryotes.

Many biological networks have to filter out useful information from a vast excess of spurious interactions. In this Letter, we use computational evolution to predict design features of networks processing ligand categorization. The important problem of early immune response is considered as a case study. Rounds of evolution with different constraints uncover elaborations of the same network motif we name "adaptive sorting." Corresponding network substructures can be identified in current models of immune recognition. Our work draws a deep analogy between immune recognition and biochemical adaptation.

Endonucleolytic cleavage within polycistronic mRNAs can lead to differential stability, and thus discordant abundance, among cotranscribed genes. RNase Y, the major endonuclease for mRNA decay in , was originally identified for its cleavage activity toward the operon, an event that differentiates the synthesis of a glycolytic enzyme from its transcriptional regulator. A three-protein Y-complex (YlbF, YmcA, and YaaT) was recently identified as also being required for this cleavage in vivo, raising the possibility that it is an accessory factor acting to regulate RNase Y. However, whether the Y-complex is broadly required for RNase Y activity is unknown. Here, we used end-enrichment RNA sequencing (Rend-seq) to globally identify operon mRNAs that undergo maturation posttranscriptionally by RNase Y and the Y-complex. We found that the Y-complex is required for the majority of RNase Y-mediated mRNA maturation events and also affects riboswitch abundance in In contrast, noncoding RNA maturation by RNase Y often does not require the Y-complex. Furthermore, deletion of RNase Y has more pleiotropic effects on the transcriptome and cell growth than deletions of the Y-complex. We propose that the Y-complex is a specificity factor for RNase Y, with evidence that its role is conserved in.