The Role of Posttranscriptional Modification in Stabilization of Transfer RNA from Hyperthermophiles

The influence of posttranscriptional modification on structural stabilization of tRNA from hyperthermophilic archaea was studied, using Pyrococcus furiosus (growth optimum 100 °C) as a primary model. Optical melting temperatures (T_m) of unfractionated tRNA in 20 mM Mg²⁺ are 97 °C for P. furiosus and 101.5 °C for Pyrodictium occultum (growth optimum, 105 °C). These values are ∼20 °C higher than predicted solely from G-C content and are attributed primarily to posttranscriptional modification. Twenty-three modified nucleosides were determined in total digests of P. furiosus tRNA by combined HPLC-mass spectrometry. From cells cultured at 70, 85, and 100 °C, progressively increased levels of modification were observed within three families of nucleosides, the most highly modified forms of which were N⁴-acetyl-2′-O-methylcytidine (ac⁴Cm), N²,N²,2′-O-trimethylguanosine [formula omitted], and 5-methyl-2-thiouridine (m⁵s²U). Nucleosides ac⁴Cm and [formula omitted], which are unique to the archaeal hyperthermophiles, were shown in earlier NMR studies to exhibit unusually high conformational stabilities that favor the C3′-endo form [Kawai, G., et al. (1991) Nucleic Acids Symp. Ser. 21, 49′50; (1992) Nucleosides Nucleotides 11, 759–771]. The sequence location of m⁵s²U was determined by mass spectrometry to be primarily at tRNA position 54, a site of known thermal stabilization in the bacterial thermophile Thermus thermophilus [Horie, N., et al. (1985) Biochemistry 24, 5711′5715]. It is concluded that selected posttranscriptional modifications in archaeal thermophiles play major stabilizing roles beyond the effects of Mg²⁺ binding and G-C content, and are proportionally more important and have evolved with greater structural diversity at the nucleoside level than in the bacterial thermophiles.