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 (Tm) of unfractionated tRNA in 20 mM Mg2+ are 97 OC for P. furiosus and 101.5 OC for Pyrodictium occultum (growth optimum, 105 "C). These values are -20 OC 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 OC, 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 (miGm), and 5-methyl-2- thiouridine (mSs2U). Nucleosides ac4Cm and miGm, which are unique to the archaeal hyperthermophiles, were shown in earlier NMR studies to exhibit unusually high conformational stabilities that favor the C3'-endoform (Kawai, G., et al. (1991) Nucleic Acids Symp. Ser. 21,49-50; (1992) Nucleosides Nucleotides 11, 759-7711. The sequence location of mSs2U 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, 57 1 1-571 51. 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.