G-Quadruplex Structures in 16S rRNA Regions Correlate with Thermal Adaptation in Prokaryotes

G-quadruplex (G4) structure is a nucleic acid secondary structure formed by sequences rich in guanine, playing essential roles in various biological processes such as gene regulation, maintenance of genome stability, and adaptation to environmental stresses. Although prokaryotes growing at high temperatures have higher GC contents, the pattern of G4 structure presence associated with GC content variation in thermal adaptation within genomes and ribosomal genes is rarely reported. In this study, we analyzed 681 bacterial genomes to investigate the role of G4 structures in thermal adaptation. Our findings revealed a strong positive correlation between G4 patterns in the region encoding 16S rRNA genes and optimal growth temperatures (Topt), whereas genomic GC content and overall G4 patterns did not show significant correlations with Topt. Evolutionary analysis showed significant differences in G4 stability between Thermotoga (Topt[≥] 80 {degrees}C) and Pseudothermotoga (60{degrees}C [≤] Topt < 80{degrees}C) species, with Thermotoga species exhibiting higher G4 stability, indicating stronger selective pressure for G4 stability under extreme conditions. Circular dichroism analysis showed that specific base mutations at key sites resulted in the absence of G4 thermal stability and structural integrity in Thermotoga compared to Pseudothermotoga. Collectively, this study suggests that the G4 structures in 16S rRNA encoding regions emerged as key indicators of thermal adaptation and contributes to our understanding of the molecular basis of thermal adaptation.