THE ROLE OF POSTTRANSCRIPTIONAL MODIFICATION IN STABILIZATION OF TRANSFER-RNA FROM HYPERTHERMOPHILES

The influence of posttranscriptional modification on structural stabil ization of tRNA from hyperthermophilic archaea was studied, using Pyro coccus furiosus (growth optimum 100 degrees C) as a primary model. Opt ical melting temperatures (T-m) of unfractionated tRNA in 20 mM Mg2+ a re 97 degrees C for P. furiosus and 101.5 degrees C for Pyrodictium oc cultum (growth optimum, 105 degrees C). These values are similar to 20 degrees C higher than predicted solely from G-C content and are attri buted primarily to posttranscriptional modification. Twenty-three modi fied nucleosides were determined in total digests of P. furiosus tRNA by combined HPLC-mass spectrometry. From cells cultured at 70, 85, and 100 degrees C, progressively increased levels of modification were ob served within three families of nucleosides, the most highly modified forms of which were N-4-acetyl-2'-O-methylcytidine (ac(4)Cm), N-2,N-2, 2'-O-trimethylguanosine (m(2)(2)Gm), and 5-methyl-2-thiouridine (m(5)S (2)U). Nucleosides ac(4)Cm and m(2)(2)Gm, which are unique to the arch aeal hyperthermophiles, were shown in earlier NMR studies to exhibit u nusually high conformational stabilities that favor the C3'-endo form [Kawai, G., et al. (1991) Nucleic Acids Symp. Ser. 21, 49-50; (1992) N ucleosides Nucleotides 11, 759-771]. The sequence location of m(5)S(2) U was determined by mass spectrometry to be primarily at tRNA position 54, a site of known thermal stabilization in the bacterial thermophil e Thermus thermophilus [Horie, N., et al. (1985) Biochemistry 24, 5711 -5715]. It is concluded that selected posttranscriptional modification s in archaeal thermophiles play major stabilizing roles beyond the eff ects of Mg2+ binding and G-C content, and are proportionally more impo rtant and have evolved with greater structural diversity at the nucleo side level than in the bacterial thermophiles.