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IntroductionThe molecular mechanisms underlying pressure adaptation remain largely unexplored, despite their significance for understanding biological adaptation and improving sterilization methods in the food and beverage industry. The heat shock response leads to a global stabilization of the proteome. Prior research suggested that the heat shock regulon may exhibit a transcriptional response to high-pressure stress. MethodsIn this study, we investigated the pressure-dependent heat shock response inE. colistrains using plasmid-borne green fluorescent protein (GFP) promoter fusions and fluorescence fluctuation microscopy. ResultsWe quantitatively confirm that key heat shock genes-rpoH,rpoE,dnaK, andgroEL- are transcriptionally upregulated following pressure shock in both piezosensitiveEscherichia coliand a more piezotolerant laboratory-evolved strain, AN62. Our quantitative imaging results provide the first single cell resolution measurements for both the heat shock and pressure shock transcriptional responses, revealing not only the magnitude of the responses, but also the biological variance involved. Moreover, our results demonstrate distinct responses in the pressure-adapted strain. Specifically,P_groELis upregulated more thanP_dnaKin AN62, while the reverse is true in the parental strain. Furthermore, unlike in the parental strain, the pressure-induced upregulation ofP_rpoEis highly stochastic in strain AN62, consistent with a strong feedback mechanism and suggesting that RpoE could act as a pressure sensor. DiscussionDespite its capacity to grow at pressures up to 62 MPa, the AN62 genome shows minimal mutations, with notable single nucleotide substitutions in genes of the transcriptionally importantbsubunit of RNA polymerase and the Rho terminator. In particular, the mutation in RNAP is one of a cluster of mutations known to confer rifampicin resistance toE. colivia modification of RNAP pausing and termination efficiency. The observed differences in the pressure and heat shock responses between the parental MG1655 strain and the pressure-adapted strain AN62 could arise in part from functional differences in their RNAP molecules. 

Conformational dynamics play essential roles in RNA function. However, detailed structural characterization of excited states of RNA remains challenging. Here, we apply high hydrostatic pressure (HP) to populate excited conformational states of tRNA Lys3 , and structurally characterize them using a combination of HP 2D-NMR, HP-SAXS (HP-small-angle X-ray scattering), and computational modeling. HP-NMR revealed that pressure disrupts the interactions of the imino protons of the uridine and guanosine U–A and G–C base pairs of tRNA Lys3 . HP-SAXS profiles showed a change in shape, but no change in overall extension of the transfer RNA (tRNA) at HP. Configurations extracted from computational ensemble modeling of HP-SAXS profiles were consistent with the NMR results, exhibiting significant disruptions to the acceptor stem, the anticodon stem, and the D-stem regions at HP. We propose that initiation of reverse transcription of HIV RNA could make use of one or more of these excited states. 

Commitment to cell division at the end of G1 phase, termed Start in the budding yeast Saccharomyces cerevisiae , is strongly influenced by nutrient availability. To identify new dominant activators of Start that might operate under different nutrient conditions, we screened a genome-wide ORF overexpression library for genes that bypass a Start arrest caused by absence of the G1 cyclin Cln3 and the transcriptional activator Bck2. We recovered a hypothetical gene YLR053c , renamed NRS1 for Nitrogen-Responsive Start regulator 1, which encodes a poorly characterized 108 amino acid microprotein. Endogenous Nrs1 was nuclear-localized, restricted to poor nitrogen conditions, induced upon TORC1 inhibition, and cell cycle-regulated with a peak at Start. NRS1 interacted genetically with SWI4 and SWI6 , which encode subunits of the main G1/S transcription factor complex SBF. Correspondingly, Nrs1 physically interacted with Swi4 and Swi6 and was localized to G1/S promoter DNA. Nrs1 exhibited inherent transactivation activity, and fusion of Nrs1 to the SBF inhibitor Whi5 was sufficient to suppress other Start defects. Nrs1 appears to be a recently evolved microprotein that rewires the G1/S transcriptional machinery under poor nitrogen conditions. 

Sampling and genomic efforts over the past decade have revealed an enormous quantity and diversity of life in Earth's extreme environments. This new knowledge of life on Earth poses the challenge of understandingits molecular basis in such inhospitable conditions, given that such conditions lead to loss of structure and of function in biomolecules from mesophiles. In this review, we discuss the physicochemical properties of extreme environments. We present the state of recent progress in extreme environmental genomics. We then present an overview of our current understanding of the biomolecular adaptation to extreme conditions. As our current and future understanding of biomolecular structure–function relationships in extremophiles requires methodologies adapted to extremes of pressure, temperature, and chemical composition, advances in instrumentation for probing biophysical properties under extreme conditions are presented. Finally, we briefly discuss possible future directions in extreme biophysics.