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

Jeffrey A. Kowalak, Joseph J. Dalluge, James A. McCloskey, Karl O. Stetter

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188 Scopus citations


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 °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.

Original languageEnglish (US)
Pages (from-to)7869-7876
Number of pages8
Issue number25
StatePublished - Jun 1 1994


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