Protein synthesis is the most energy-consuming process in a proliferating cell, and understanding what controls protein abundances represents a key question in biology and biotechnology. We quantified absolute abundances of 5,354 mRNAs and 2,198 proteins in Saccharomyces cerevisiae under ten environmental conditions and protein turnover for 1,384 proteins under a reference condition. The overall correlation between mRNA and protein abundances across all conditions was low (0.46), but for differentially expressed proteins (n = 202), the median mRNA-protein correlation was 0.88. We used these data to model translation efficiencies and found that they vary more than 400-fold between genes. Non-linear regression analysis detected that mRNA abundance and translation elongation were the dominant factors controlling protein synthesis, explaining 61% and 15% of its variance. Metabolic flux balance analysis further showed that only mitochondrial fluxes were positively associated with changes at the transcript level. The present dataset represents a crucial expansion to the current resources for future studies on yeast physiology.

Persistence is a reversible and low-frequency phenomenon allowing a subpopulation of a clonal bacterial population to survive antibiotic treatments. Upon removal of the antibiotic, persister cells resume growth and give rise to viable progeny. Type II toxin-antitoxin (TA) systems were assumed to play a key role in the formation of persister cells in Escherichia coli based on the observation that successive deletions of TA systems decreased persistence frequency. In addition, the model proposed that stochastic fluctuations of (p)ppGpp levels are the basis for triggering activation of TA systems. Cells in which TA systems are activated are thought to enter a dormancy state and therefore survive the antibiotic treatment. Using independently constructed strains and newly designed fluorescent reporters, we reassessed the roles of TA modules in persistence both at the population and single-cell levels. Our data confirm that the deletion of 10 TA systems does not affect persistence to ofloxacin or ampicillin. Moreover, microfluidic experiments performed with a strain reporting the induction of the yefM-yoeB TA system allowed the observation of a small number of type II persister cells that resume growth after removal of ampicillin. However, we were unable to establish a correlation between high fluorescence and persistence, since the fluorescence of persister cells was comparable to that of the bulk of the population and none of the cells showing high fluorescence were able to resume growth upon removal of the antibiotic. Altogether, these data show that there is no direct link between induction of TA systems and persistence to antibiotics.

Export of macromolecules via extracellular membrane-derived vesicles (MVs) plays an important role in the biology of Gram-negative bacteria. Gram-positive bacteria have also recently been reported to produce MVs; however, the composition and mechanisms governing vesiculogenesis in Gram-positive bacteria remain undefined. Here, we describe MV production in the Gram-positive human pathogen group A streptococcus (GAS), the etiological agent of necrotizing fasciitis and streptococcal toxic shock syndrome. M1 serotype GAS isolates in culture exhibit MV structures both on the cell wall surface and in the near vicinity of bacterial cells. A comprehensive analysis of MV proteins identified both virulence-associated protein substrates of the general secretory pathway in addition to “anchorless surface proteins.” Characteristic differences in the contents, distributions, and fatty acid compositions of specific lipids between MVs and GAS cell membrane were also observed. Furthermore, deep RNA sequencing of vesicular RNAs revealed that GAS MVs contained differentially abundant RNA species relative to bacterial cellular RNA. MV production by GAS strains varied in a manner dependent on an intact two-component system, CovRS, with MV production negatively regulated by the system. Modulation of MV production through CovRS was found to be independent of both GAS cysteine protease SpeB and capsule biosynthesis. Our data provide an explanation for GAS secretion of macromolecules, including RNAs, lipids, and proteins, and illustrate a regulatory mechanism coordinating this secretory response.

The Gcn pathway is conserved in all eukaryotes, including mammals such as humans, where it is a crucial part of the integrated stress response (ISR). Gcn1 serves as an essential effector protein for the kinase Gcn2, which in turn is activated by stalled ribosomes, leading to phosphorylation of eIF2 and a subsequent global repression of translation. The fine-tuning of this adaptive response is performed by the Rbg2/Gir2 complex, a negative regulator of Gcn2. Despite the wealth of available biochemical data, information on structures of Gcn proteins on the ribosome has remained elusive. Here we present a cryo-electron microscopy structure of the yeast Gcn1 protein in complex with stalled and colliding 80S ribosomes. Gcn1 interacts with both 80S ribosomes within the disome, such that the Gcn1 HEAT repeats span from the P-stalk region on the colliding ribosome to the P-stalk and the A-site region of the lead ribosome. The lead ribosome is stalled in a nonrotated state with peptidyl-tRNA in the A-site, uncharged tRNA in the P-site, eIF5A in the E-site, and Rbg2/Gir2 in the A-site factor binding region. By contrast, the colliding ribosome adopts a rotated state with peptidyl-tRNA in a hybrid A/P-site, uncharged-tRNA in the P/E-site, and Mbf1 bound adjacent to the mRNA entry channel on the 40S subunit. Collectively, our findings reveal the interaction mode of the Gcn2-activating protein Gcn1 with colliding ribosomes and provide insight into the regulation of Gcn2 activation. The binding of Gcn1 to a disome has important implications not only for the Gcn2-activated ISR, but also for the general ribosome-associated quality control pathways.

No abstract