Interestingly, all the hyperthermophiles and thermophiles (except three variants) always grouped together, whereas the mesophiles and the psychrophiles preferred to remain in a separate cluster. Similar results were observed even in the case of k-means clustering. To demonstrate

the effect of temperature on folding patterns, k-means clustering was also performed at 20, 37 and 70 °C, which are the representative temperatures for psychrophiles, mesophiles Talazoparib clinical trial and thermophiles, respectively, using both dG and Tm values. At 20 °C, two distinct clusters were formed by the thermophiles and hyperthermophiles, whereas some of the thermophiles strayed into the groups of mesophiles and psychrophiles. At 37 °C, the thermophiles and hyperthermophiles showed a better composure and this was even strengthened further at 70 °C (Supporting Dabrafenib datasheet Information, Figs S1 and S2). Thus, tRNA folding patterns can, in principle, distinguish the organisms into groups based on their OGT. The present analysis indicates that adaptation of thermophiles and hyperthermophiles to elevated temperatures

imposes selective constraints on the number and distribution of tRNAs, the GC content of the tRNA genes and on their secondary structures and folding patterns. The reliability of nucleic acids is threatened at high temperatures either by strand separation or by chemical damage of the nucleotide constituents or at the extreme by breakage of backbone phosphodiester bonds (Grogan, 1998; Daniel & Cowan, 2000). Thus, a possible adaptation mechanism of nucleic Terminal deoxynucleotidyl transferase acids to thermophilic or hyperthermophilic conditions would be an increase in the GC content. Previous studies have shown, and our analysis with a bunch of thermophilic, hyperthermophilic, mesophilic and psychrophilic genomes confirm, that there is a strong positive correlation between the GC content of the tRNAs with OGT (r=0.85, P<0.00001). On the contrary, the GC content of genomic DNA far less

correlated with the growth temperature (r=0.25, P=0.05). However, a strong positive correlation has also been found between the GC content of rRNA with that of OGT for the organisms chosen for the present study (r=0.868, P<0.00001), suggesting that rRNA correlate better with tRNA than with the genomic DNA. One explanation could be that cellular DNA is in a topologically closed conformation, and denaturation will not result in two independent single-stranded molecules, but in a random-coiled structure with interwined strands (Marguet & Forterre, 2001). As a result, topologically closed DNA is much resistant to denaturation compared with open conformation. The tRNA molecules are not permanently integrated into larger macromolecular complexes. Therefore, in adapting to high temperatures, they must have developed mechanisms for intrinsic stabilization. Part of the stabilization energy may originate from an increased GC content.