Two Cold‐Shock Proteins Characterised as <scp>RNA</scp> Chaperone of Hyperthermophilic Archaeon <scp> <i>Pyrococcus yayanosii</i> </scp>

Cold shock proteins (Csps) play a crucial role in facilitating cellular growth at suboptimal temperatures. In this study, we identified and characterised two Csps, PyCsp and PyTRAM, in the hyperthermophilic archaeon Pyrococcus yayanosii A1. Using bio-layer interferometry (BLI) and molecular beacon assays, we demonstrated that both proteins exhibit RNA binding and unfolding activities in vitro. Heterologously expressed PyCsp and PyTRAM exhibited transcription anti-termination activity in Escherichia coli RL211 and could restore the growth of the cold-sensitive E. coli BX04 at 22°C. Knockout of the coding genes of either PyCsp or PyTRAM impaired the growth of P. yayanosii A1 at 85°C, a comparatively lower temperature to the optimal 95°C. Gene knockout and cross-complementation analyses of the coding genes for these two proteins suggest that PyCsp and PyTRAM functionally complement each other at low temperatures. PyTRAM contains the conserved TRAM domain, which is a typical characteristic of archaeal RNA chaperones. Notably, PyCsp shows low similarity to known archaeal RNA chaperones. Deletion of PYCH_0765, the gene encoding PyCsp, led to 27.5% changes in the transcriptome. This work highlights PyCsp as a non-TRAM class RNA chaperone that globally alters the transcriptome of P. yayanosii under cold shock conditions.