Abstract: Abstract Extreme thermophiles produce unusually long polyamines, including the linear caldopentamine (Cdp) and the branched pentamine tetrakis(3-aminopropyl)-ammonium (Taa), the latter containing a central quaternary ammonium moiety. Here, we compare the interaction of these two pentamines with RNA by studying the heat denaturation, the behavior in electrophoresis, and the ability of tRNA to be methylated in vitro by purified tRNA methyltransferases under various salt conditions. At concentrations in the micromolar range, the branched Taa causes a considerable increase of the T(m) of yeast tRNA(Phe) transcript by over 20 degrees C, significantly higher than stabilization by the linear counterpart Cdp. In non-denaturing gel electrophoresis, a strong and specific binding of branched Taa, but not of linear Cdp, to tRNA(Phe) was clearly evident. In both types of experiments, polyamines and monovalent metal ions compete with each other for binding sites. Structural probing showed no significant conformational changes of the tRNA upon Taa binding. In post-transcriptional in vitro methylation reactions, formation of m(2)G/m(2)(2)G by the methyltransferase Trm1p and of m(1)A by TrmIp were unaffected or slightly stimulated by polyamines. In contrast, Taa specifically inhibited Trm4p-dependent formation of m(5)C only in tRNA(Phe), likely by occupying sites that are relevant to RNA recognition by the methyltransferase.