UCSF

Protein synthesis is the most energetically expensive process in prokaryotes. Understanding how protein synthesis is regulated is critical both for decoding natural systems and for engineering synthetic protein synthesis. Protein synthesis in prokaryotes occurs on mRNAs organized into operons consisting of discrete open reading frames (ORFs) that are differentially translated by as much as 100- fold. We have applied ribosome profiling, which enables the quantitative determination of the rates of protein synthesis genome-wide in E. coli, to understand the rules that guide these differential rates of protein synthesis. We then combined ribosome profiling with DMS-seq , which monitors mRNA structure genome-wide, to monitor the relationship between mRNA structure and translation on endogenous messages genome-wide in vivo, enabling us to understand the mRNA features that instruct translation efficiencies.

We find precisely tuned synthesis rates for a wide variety of proteins --members of multi-protein complexes are made in proportion to their stoichiometry, and components of functional modules are produced differentially according to their hierarchical role. Additionally, several principles of design optimization emerge from the absolute copy number measurements. These include how the distribution of levels of different transcription factors is optimized to enable rapid responses and how a metabolic pathway (methionine biosynthesis) balances the cost of enzyme production with the requirement for its activity.

Structural probing of mRNAs reveals that operon mRNAs are organized into structural domains divided by ORF boundaries. This modular mRNA structure, rather than Shine-Dalgarno strength, specifies ORF translation efficiency. Upon cold shock, mRNA structure increases and translation decreases, but both are restored by massive induction of the Cold Shock Proteins (Csps). Csps modulate global mRNA structure and autoregulate their expression via an RNA element cued to the cellular environment, enabling mRNA structure surveillance both at cold and normal growth temperatures. Operons and Csps are present in all bacteria, suggesting that the organization of operonic mRNA structure and its surveillance system we describe are universally used to set and maintain translation.

Together, this work indicates protein synthesis is precisely controlled in prokaryotes, and this precise control requires mRNA structures designed to reflect synthesis rates. This lays the framework for both future efforts to computationally determine complex stoichiometry, and for computational design of protein synthesis rates.