||The influence of posttranscriptional modifications on structural stability, flexibility, and temperature adaptation in RNA, has been studied using the hyperthermophilic archaeon, P. furiosus and three psychrophilic bacteria, Vibrio sp. strain ANT-300, strain no. 29-6, and strain no. 5710, as primary models. Conformational stability of RNA in hyperthermophilic archaea was studied by measuring optical melting temperatures (T m]) of unfractionated tRNA from two hyperthermophilic archaea in the presence and absence of 20 mM Mg2+. T m values for tRNA from these organisms were ~ 20°C higher than predicted solely from G-C content, and are attributed primarily to posttranscriptional modification playing major stabilizing roles beyond the effects of Mg[2+ binding and G-C content. A method for the quantitation of 5,6-dihydrouridine in RNA using isotope dilution liquid chromatography-mass spectrometry is presented. To understand further the structural role for dihydrouridine in RNA, the solution conformations of Dp and ApDpA were analyzed by NMR spectroscopy and compared with those of the related uridine-containing compounds. The analyses indicate dihydrouridine destabilizes the C3'-endo sugar conformation associated with base-stacked, ordered, A-type RNA. Stabilization of the C2'-endo form is enhanced at low temperatures, indicating C2'-endo is the thermodynamically favored conformation for dihydrouridine. By promoting the flexible C2'-endo sugar conformation, posttranscriptional formation of dihydrouridine allows greater conformational flexibility in regions of RNA where tertiary interactions and loop formation must be simultaneously accommodated. Nucleoside modification has been studied in unfractionated tRNA from three psychrophilic bacteria and one psychrotrophic bacterium. Transfer RNA from these organisms is significantly hypomodified compared to that of mesophiles and thermophiles. The method described above was used to quantify dihydrouridine in tRNA from these bacteria. Transfer RNA from the psychrophiles contains 40-70% more dihydrouridine on average than tRNA from the mesophile E. coli, or the psychrotroph L. bavaricus. This finding supports the role of dihydrouridine in providing local conformational flexibility to RNA, and implicates D in structural adaptation of RNA to cold temperature, opposite to modified nucleosides in thermophilic bacteria and archaea which provide structural stability to RNA for adaptation to high temperature.