SummaryCell-autonomous circadian rhythms allow organisms to temporally orchestrate their internal state to anticipate and/or resonate with the external environment [1, 2]. Although ∼24-hr periodicity is observed across aerobic eukaryotes, the central mechanism has been hard to dissect because few simple models exist, and known clock proteins are not conserved across phylogenetic kingdoms [1, 3, 4]. In contrast, contributions to circadian rhythmicity made by a handful of post-translational mechanisms, such as phosphorylation of clock proteins by casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3), appear conserved among phyla [3, 5]. These kinases have many other essential cellular functions and are better conserved in their contribution to timekeeping than any of the clock proteins they phosphorylate [6]. Rhythmic oscillations in cellular redox state are another universal feature of circadian timekeeping, e.g., over-oxidation cycles of abundant peroxiredoxin proteins [7–9]. Here, we use comparative chronobiology to distinguish fundamental clock mechanisms from species and/or tissue-specific adaptations and thereby identify features shared between circadian rhythms in mammalian cells and non-circadian temperature-compensated respiratory oscillations in budding yeast [10]. We find that both types of oscillations are coupled with the cell division cycle, exhibit period determination by CK1 and GSK3, and have peroxiredoxin over-oxidation cycles. We also explore how peroxiredoxins contribute to YROs. Our data point to common mechanisms underlying both YROs and circadian rhythms and suggest two interpretations: either certain biochemical systems are simply permissive for cellular oscillations (with frequencies from hours to days) or this commonality arose via divergence from an ancestral cellular clock.

Genetic code expansion via amber stop suppression provides a powerful tool for introducing non-proteinogenic functionalities into proteins for a broad range of applications. However, ribosomal incorporation of non-canonical amino acids (ncAA) by means of engineered AARS often proceeds with significantly reduced efficiency compared to sense codon translation. Here, we report the implementation of a versatile platform for the development of engineered aminoacyl-tRNA synthetases with enhanced efficiency in mediating ncAA incorporation via amber stop codon suppression. This system integrates a white/blue colony screen with a plate-based colorimetric assay, thereby combining high-throughput capabilities with reliable and quantitative measurement of AARS-dependent ncAA incorporation efficiency. This two-tier functional screening system was successfully applied to obtain a pyrrolysyl-tRNA synthetase variant (CrtK-RS(4.1)) with significantly improved efficiency (+250-370%) toward mediating the incorporation of Nε-crotonyl-lysine and other lysine analogs of relevance for the study of protein post-translational modifications, into a target protein. Interestingly, the beneficial mutations accumulated by CrtK-RS(4.1) were found to localize within the non-catalytic N-terminal domain of the enzyme, and could be transferred to another PylRS variant improving its ability to incorporate its corresponding ncAA substrate. This work introduces and validates an efficient platform for the improvement of AARSs that could be readily extended to other members of this enzyme family and/or other target non-canonical amino acids.

No abstract

It has been more than a decade since the last comprehensive review of the phase-variable uropathogen-associated pyelonephritis-associated pilus (pap) genetic switch. Since then, important data have come to light, including additional factors that regulate pap expression, better characterization of H-NS regulation, the structure of the Lrp octamer in complex with pap regulatory DNA, the temperature-insensitive phenotype of a mutant lacking the acetyltransferase RimJ, evidence that key components of the regulatory machinery are acetylated, and new insights into the role of DNA binding by key regulators in shaping both the physical structure and regulatory state of the papI and papBA promoters.

Leucine-responsive regulatory protein (Lrp) is one of the most impactful regulators in E. coli and other bacteria. Lrp senses nutrient conditions and responds by controlling strategies for virulence, cellular motility, and nutrient acquisition.