In nature, bacteria frequently experience many adverse conditions, including heat, oxidation, acidity, and hyperosmolarity, which all tend to slow down if not outright stop cell growth. Previous work on bacterial stress mainly focused on understanding gene regulatory responses. Much less is known about how stresses compromise protein synthesis, which is the major driver of cell growth. Here, we quantitatively characterize the translational capacity of Escherichia coli cells growing exponentially under hyperosmotic stress. We found that hyperosmotic stress affects bacterial protein synthesis through reduction of the translational elongation rate, which is largely compensated for by an increase in the cellular ribosome content compared with nutrient limitation at a similar growth rate. The slowdown of translational elongation is attributed to a reduction in the rate of binding of tRNA ternary complexes to the ribosomes.IMPORTANCE Hyperosmotic stress is a common stress condition confronted by E. coli during infection of the urinary tract. It can significantly compromise the bacterial growth rate. Protein translation capacity is a critical component of bacterial growth. In this study, we find for the first time that hyperosmotic stress causes substantial slowdown in bacterial ribosome translation elongation. The slowdown of translation elongation originates from a reduced binding rate of tRNA ternary complex to the ribosomes.

Bacteria growing under different conditions experience a broad range of demand on the rate of protein synthesis, which profoundly affects cellular resource allocation. During fast growth, protein synthesis has long been known to be modulated by adjusting the ribosome content, with the vast majority of ribosomes engaged at a near-maximal rate of elongation. Here, we systematically characterize protein synthesis by Escherichia coli, focusing on slow-growth conditions. We establish that the translational elongation rate decreases as growth slows, exhibiting a Michaelis-Menten dependence on the abundance of the cellular translational apparatus. However, an appreciable elongation rate is maintained even towards zero growth, including the stationary phase. This maintenance, critical for timely protein synthesis in harsh environments, is accompanied by a drastic reduction in the fraction of active ribosomes. Interestingly, well-known antibiotics such as chloramphenicol also cause a substantial reduction in the pool of active ribosomes, instead of slowing down translational elongation as commonly thought.

A detailed understanding of tRNA/mRNA translocation requires measurement of the forces generated by the ribosome during this movement. Such measurements have so far remained elusive and, thus, little is known about the relation between force and translocation and how this reflects on its mechanism and regulation. Here, we address these questions using optical tweezers to follow translation by individual ribosomes along single mRNA molecules, against an applied force. We find that translocation rates depend exponentially on the force, with a characteristic distance close to the one-codon step, ruling out the existence of sub-steps and showing that the ribosome likely functions as a Brownian ratchet. We show that the ribosome generates ∼13 pN of force, barely sufficient to unwind the most stable structures in mRNAs, thus providing a basis for their regulatory role. Our assay opens the way to characterizing the ribosome's full mechano-chemical cycle.

Editing domains of aminoacyl tRNA synthetases correct tRNA charging errors to maintain translational fidelity. A mutation in the editing domain of alanyl tRNA synthetase (AlaRS) in Aars ^sti mutant mice results in an increase in the production of serine-mischarged tRNA^Ala and the degeneration of cerebellar Purkinje cells. Here, using positional cloning, we identified Ankrd16, a gene that acts epistatically with the Aars ^sti mutation to attenuate neurodegeneration. ANKRD16, a vertebrate-specific protein that contains ankyrin repeats, binds directly to the catalytic domain of AlaRS. Serine that is misactivated by AlaRS is captured by the lysine side chains of ANKRD16, which prevents the charging of serine adenylates to tRNA^Ala and precludes serine misincorporation in nascent peptides. The deletion of Ankrd16 in the brains of Aars^sti/sti mice causes widespread protein aggregation and neuron loss. These results identify an amino-acid-accepting co-regulator of tRNA synthetase editing as a new layer of the machinery that is essential to the prevention of severe pathologies that arise from defects in editing.