Chazan, A. | Search bioRxiv

bioRxiv2021

DNA dynamics dictates p53 functional outcome

The tumor suppressor protein p53 is situated in the midst of a complex network that is activated in response to cellular stress. An unresolved question is how p53 activates its myriad target genes in response to the severity of the stress signal and consequently coordinates the functional outcome in a timely manner. We have previously shown that DNA torsional flexibility distinguishes among p53 response elements (REs). Here we calculated the flexibility of over 200 p53 REs. By connecting functional pathways of p53-dependent genes to the calculated flexibility of their REs, we show that genes belonging to pathways activated rapidly upon stress (e.g., cell-cycle arrest, energy metabolism and innate immunity) contain REs that are significantly more flexible relative to REs of genes involved in pathways that need to be more strictly regulated or are activated later in the response to stress (e.g., intrinsic apoptosis and p53 negative regulation). The global structural properties of several p53 REs belonging to the different pathways were experimentally validated. Additionally, reporter gene expression driven by flexible p53 REs occurred at lower p53 levels and with faster rates than expression from rigid REs. Moreover, analysis of published endogenous mRNA levels of p53 target genes as a function of the flexibility of their REs support our hypothesis. Overall, we demonstrate that DNA flexibility of p53 REs contributes significantly to the timely expression of p53 target genes and thereby plays an important role in cell-faith decisions in the p53 circuity.

biochemistry↗

bioRxiv2021

Characterization of heliorhodopsins detected via functional metagenomics in freshwater Actinobacteria, Chloroflexi and Archaea

Rhodopsins are widespread in microbes residing in diverse aquatic environments across the globe. Recently, a new unusual rhodopsin family, the heliorhodopsins (HeRs), was discovered, distributed among diverse bacteria, archaea, eukarya and even viruses. Here, using functional metagenomics on samples from Lake HaHula and Ein Afek reserve, we found and characterized ten HeRs representing divergent members of the family. The expressed HeRs absorb light in the green and yellow wavelengths and originate from Actinobacteria, Chloroflexi and Archaea. The photocycle of the HeR from Chloroflexi revealed a low accumulation of the M-intermediate that we connect to the lack of two conserved histidine residues in transmembrane helices 1 and 2 in this protein. Another of HeR, from Actinobacteria, exhibited an unusually fast photocycle (166 ms, 5 times faster than HeR-48C12). To further explore the still unresolved question of the HeR function, we performed an analysis of protein families among genes neighboring HeRs, in our clones and thousands of other microbes. This analysis revealed a putative connection between HeRs and genes involved in oxidative stress. At the same time, very few protein families were found to distinguish genes surrounding prokaryotic HeRs from those surrounding rhodopsin pumps. The strongest association was found with the DegV family involved in activation of fatty acids and uncharacterized family DUF2177, which allowed us to hypothesize that HeRs are involved in membrane lipid remodeling. This work further establishes functional metagenomics as a simple and fruitful method of screening for new rhodopsins. SignificanceThe recently discovered divergent rhodopsin family of heliorhodopsins is abundant in freshwater environments. In this study, we sampled a habitat rich in dissolved organic matter to increase our chances of finding spectrally shifted rhodopsins. Using functional metagenomics, diverse heliorhodopsins absorbing green and yellow light were discovered. The metagenomic clones originated from diverse prokaryotic groups: Actinobacteria, Chloroflexi and even Archaea, emphasizing the versatility of the E. coli expression system used. Photocycles of representative heliorhodopsins were measured and exhibited diverse kinetic characteristics. Analysis of genes neighboring heliorhodopsins in diverse prokaryotes revealed their putative connection to membrane lipid re-modeling and oxidative stress. Our findings suggest that functional metagenomics is a productive method for the discovery of new and diverse rhodopsins.

microbiology↗