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Abstract Chaperones are essential to the co-translational folding of most proteins. However, the principles of co-translational chaperone interaction throughout the proteome are poorly understood, as current methods are restricted to few substrates and cannot capture nascent protein folding or chaperone binding sites, precluding a comprehensive understanding of productive and erroneous protein biosynthesis. Here, by integrating genome-wide selective ribosome profiling, single-molecule tools, and computational predictions using AlphaFold we show that the binding of the mainE. colichaperones involved in co-translational folding, Trigger Factor (TF) and DnaK correlates with “unsatisfied residues” exposed on nascent partial folds – residues that have begun to form tertiary structure but cannot yet form all native contacts due to ongoing translation. This general principle allows us to predict their co-translational binding across the proteome based on sequence only, which we verify experimentally. The results show that TF and DnaK stably bind partially folded rather than unfolded conformers. They also indicate a synergistic action of TF guiding intra-domain folding and DnaK preventing premature inter-domain contacts, and reveal robustness in the larger chaperone network (TF, DnaK, GroEL). Given the complexity of translation, folding, and chaperone functions, our predictions based on general chaperone binding rules indicate an unexpected underlying simplicity.
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This content will become publicly available on November 18, 2025
Response and adaptation of the transcriptional heat shock response to pressure
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,PgroELis upregulated more thanPdnaKin AN62, while the reverse is true in the parental strain. Furthermore, unlike in the parental strain, the pressure-induced upregulation ofPrpoEis 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.
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- Award ID(s):
- 2019455
- PAR ID:
- 10616190
- Publisher / Repository:
- frontiersin.org
- Date Published:
- Journal Name:
- Frontiers in Microbiology
- Volume:
- 15
- ISSN:
- 1664-302X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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