An evolutionarily conserved phosphoserine-arginine salt bridge in the interface between ribosomal proteins uS4 and uS5 regulates translational accuracy in Saccharomyces cerevisiae.

Protein-protein and protein-rRNA interactions at the interface between ribosomal proteins uS4 and uS5 are thought to maintain the accuracy of protein synthesis by increasing selection of cognate aminoacyl-tRNAs. Selection involves a major conformational change-domain closure-that stabilizes aminoacyl-tRNA in the ribosomal acceptor (A) site. This has been thought a constitutive function of the ribosome ensuring consistent accuracy. Recently, the Saccharomyces cerevisiae Ctk1 cyclin-dependent kinase was demonstrated to ensure translational accuracy and Ser238 of uS5 proposed as its target. Surprisingly, Ser238 is outside the uS4-uS5 interface and no obvious mechanism has been proposed to explain its role. We show that the true target of Ctk1 regulation is another uS5 residue, Ser176, which lies in the interface opposite to Arg57 of uS4. Based on site specific mutagenesis, we propose that phospho-Ser176 forms a salt bridge with Arg57, which should increase selectivity by strengthening the interface. Genetic data show that Ctk1 regulates accuracy indirectly; the data suggest that the kinase Ypk2 directly phosphorylates Ser176. A second kinase pathway involving TORC1 and Pkc1 can inhibit this effect. The level of accuracy appears to depend on competitive action of these two pathways to regulate the level of Ser176 phosphorylation.

Engineered Biosensors in an Encapsulated and Deployable System for Environmental Chemical Detection.

The long-term exposure of low levels of the fungicide, 2-phenylphenol (2-PP), to the environment presents a hazard to human and aquatic health. The cost and difficulty in large-scale production limit the use of existing sensors to detect 2-PP for applications such as personal protection and persistent environmental monitoring of large areas. While advances have been made in using whole cells as biosensors for specific chemical detection, a whole-cell biosensor system with robust biocontainment for field deployment and a strong visual reporter for readouts in the deployed environment has yet to be realized. Here, engineered biosensors in an encapsulated and deployable system (eBEADS) were created to demonstrate a portable, no-power living sensor for detection of 2-PP in the environment. A whole-cell living sensor to detect 2-PP was developed in Escherichia coli by utilizing the 2-PP degradation pathway with an agenetic amplification circuit to produce a visual colorimetric output. To enable field deployment, a physical biocontainment system comprising polyacrylamide alginate beads was designed to encapsulate sensor strains, support long-term viability without supplemental nutrients, and allow permeability of the target analyte. Integration of materials and sensing strains has led to the development of a potential deployable end-to-end living sensor that, with the addition of an amplification circuit, has up to a 66-fold increase in ß-galactosidase reporter output over non-amplified strains, responding to as little as 1 µM 2-PP while unencapsulated and 10 µM 2-PP while encapsulated. eBEADS enable sensitive and specific in-field detection of environmental perturbations and chemical threats without electronics.