Artificial cells capable of both sensing and sending chemical messages to bacteria have yet to be built. Here we show that artificial cells that are able to sense and synthesize quorum signaling molecules can chemically communicate with V. fischeri, V. harveyi, E. coli, and P. aeruginosa. Activity was assessed by fluorescence, luminescence, RT-qPCR, and RNA-seq. Two potential applications for this technology were demonstrated. First, the extent to which artificial cells could imitate natural cells was quantified by a type of cellular Turing test. Artificial cells capable of sensing and in response synthesizing and releasing N-3-(oxohexanoyl)homoserine lactone showed a high degree of likeness to natural V. fischeri under specific test conditions. Second, artificial cells that sensed V. fischeri and in response degraded a quorum signaling molecule of P. aeruginosa (N-(3-oxododecanoyl)homoserine lactone) were constructed, laying the foundation for future technologies that control complex networks of natural cells.

No abstract

Although RNA synthesis can be reliably controlled with different T7 transcriptional promoters during cell-free gene expression with the PURE system, protein synthesis remains largely unaffected. To better control protein levels, we investigated a series of ribosome binding sites (RBSs). Although RBS strength did strongly affect protein synthesis, the RBS sequence could explain less than half of the variability of the data. Protein expression was found to depend on other factors besides the strength of the RBS, including the GC content of the coding sequence. The complexity of protein synthesis in comparison to RNA synthesis was observed by the higher degree of variability associated with protein expression. This variability was also observed in an E. coli cell extract-based system. However, the coefficient of variation was larger with E. coli RNA polymerase than with T7 RNA polymerase, consistent with the increased complexity of E. coli RNA polymerase.

Chemical communication is ubiquitous in biology, and so efforts in building convincing cellular mimics must consider how cells behave on a population level. Simple model systems have been built in the laboratory that show communication between different artificial cells and artificial cells with natural, living cells. Examples include artificial cells that depend on purely abiological components and artificial cells built from biological components and are driven by biological mechanisms. However, an artificial cell solely built to communicate chemically without carrying the machinery needed for self-preservation cannot remain active for long periods of time. What is needed is to begin integrating the pathways required for chemical communication with metabolic-like chemistry so that robust artificial systems can be built that better inform biology and aid in the generation of new technologies.

Yra1 is an mRNA export adaptor involved in mRNA biogenesis and export in S . cerevisiae . Yra1 overexpression was recently shown to promote accumulation of DNA:RNA hybrids favoring DNA double strand breaks (DSB), cell senescence and telomere shortening, via an unknown mechanism. Yra1 was also identified at an HO-induced DSB and Yra1 depletion causes defects in DSB repair. Previous work from our laboratory showed that Yra1 ubiquitination by Tom1 is important for mRNA export. Here, we found that Yra1 is also ubiquitinated by the SUMO-targeted ubiquitin ligases Slx5-Slx8 implicated in the interaction of irreparable DSB with nuclear pores. We further show that Yra1 binds an HO-induced irreparable DSB in a process dependent on resection. Importantly, a Yra1 mutant lacking the evolutionarily conserved C-box is not recruited to an HO-induced irreparable DSB and becomes lethal under DSB induction in a HO-cut reparable system. Together, the data provide evidence that Yra1 plays a crucial role in DSB repair via homologous recombination. While Yra1 sumoylation and/or ubiquitination are dispensable, the Yra1 C-box region is essential in this process.

Equal contribution + Corresponding author: Francoise.Stutz@unige.ch 1 ABSTRACT (168 words) 2 3Yra1 is an mRNA export adaptor involved in mRNA biogenesis and 4 export in S. cerevisiae. Yra1 overexpression was recently shown to promote 5 accumulation of DNA:RNA hybrids favoring DNA double strand breaks (DSB), 6 cell senescence and telomere shortening, via an unknown mechanism. Yra1 7 was also identified at an HO-induced DSB and Yra1 depletion causes defects 8 in DSB repair. Previous work from our laboratory showed that Yra1 9 ubiquitination by Tom1 is important for mRNA export. Interestingly, we found 10 that Yra1 is also ubiquitinated by the SUMO-targeted ubiquitin ligases Slx5-11 Slx8 implicated in the interaction of irreparable DSB with nuclear pores. Here 12 we show that Yra1 binds an HO-induced irreparable DSB. Importantly, a Yra1 13 mutant lacking the evolutionarily conserved C-box is not recruited to an HO-14 induced irreparable DSB and becomes lethal under DSB induction in a HO-15 cut reparable system. Together, the data provide evidence that Yra1 plays a 16 crucial role in DSB repair via homologous recombination. Unexpectedly, while 17 the Yra1 C-box is essential, Yra1 sumoylation and/or ubiquitination are 18 dispensable in this process. 19 20 21 Keywords: Yra1, HO endonuclease cut, genome instability, DSB repair, 22 homologous recombination. 49 export defect although this domain is not implicated in Mex67 interaction nor 50 RNA binding in vitro, suggesting that it may contribute to Yra1 function by 51 ensuring optimal folding of the protein. Loss of the highly conserved Yra1 C-52 box (yra1(1-210) mutant) does not cause an obvious poly(A)+ mRNA export 53 defect, but it is required for optimal growth (7). This observation is consistent 54 with the fact that the C-box does not play a major role in Mex67 or RNA 55 binding and suggests that this highly conserved 16 amino acids sequence 56 may be important for another aspect of Yra1 function. 57 Different layers of regulations have been shown to modulate Yra1 levels 58 and function in mRNA biogenesis. We have previously shown that Yra1 59 ubiquitination by the E3 ligase Tom1 displaces Yra1 from messenger 60 ribonucleoparticles (mRNPs) as a quality control signal for correctly processed 61 mRNP prior to export into the cytoplasm (8). Another important feature for Yra1 62 regulation is that the YRA1 gene harbors the second largest intron (776 nt) in 63 the S. cerevisiae genome, containing a non-canonical branchpoint sequence 64 (BS, gACUAAC) after a long first exon (300 nt). An excess of Yra1 protein 65 prevents YRA1 pre-mRNA splicing and promotes export of the unspliced 66 transcript into the cytoplasm where it is degraded by the 5' to 3' decay pathway 67 ( 4, 9). It has been reported previously that the yra1∆intron mutant shows Yra1 68 protein overexpression (7) that is toxic for cell growth (10, 11) and impairs 69 poly(A)+ RNA export (12, 13). The presence of the YRA1 intron is important to 70 maintain optimal Yra1 protein levels through Yra1 auto-regulation a...

Wet–dry cycles driven by heating to high temperatures are frequently invoked for the prebiotic synthesis of peptides. Similarly, iron–sulfur clusters are often cited as an example of an ancient catalyst that helped prune early chemical systems into metabolic-like pathways. Because extant iron–sulfur clusters are metallocofactors of protein enzymes and nearly ubiquitous across biology, a reasonable hypothesis is that prebiotic iron–sulfur peptides formed on the early Earth. However, iron–sulfur clusters are coordinated by multiple cysteine residues, and the stability of cysteines to the heat steps of wet–dry cycles has not been determined. It, therefore, has remained unclear if the peptides needed to stabilize the formation of iron–sulfur clusters could have formed. If not, then iron–sulfur-dependent activity may have emerged later, when milder, more biological-like peptide synthesis machinery took hold. Here, we report the thermal stability of cysteine-containing peptides. We show that temperatures of 150 °C lead to the rapid degradation of cysteinyl peptides. However, the presence of Mg2+ at environmentally reasonable concentrations leads to significant protection. Thiophilic metal ions also protect against degradation at 150 °C but require concentrations not frequently observed in the environment. Nevertheless, cysteine-containing peptides are stable at lower, prebiotically plausible temperatures in seawater, carbonate lake, and ferrous lake conditions. The data are consistent with the persistence of cysteine-containing peptides on the early Earth in environments rich in metal ions. High concentrations of Mg2+ are common intra- and extra-cellularly, suggesting that the protection afforded by Mg2+ may reflect conditions that were present on the prebiotic Earth.