Effect of a quaternary pentamine on RNA stabilization and enzymatic methylation

Abstract

AbstractExtreme thermophiles produce unusually long polyamines, including the linear caldopentamine (Cdp) and the branched pentamine tetrakis(3-aminopropyl)-ammonium (Taa), with the latter containing a central quaternary ammonium moiety. Here we compare the interaction of these two pentamines with RNA by studying the heat denaturation, electrophoretic behavior, and ability of tRNA to be methylatedin vitroby purified tRNA methyltransferases under various salt conditions. At concentrations in the micromolar range, branched Taa causes a considerable increase in the melting temperature (Tm) of yeast tRNAPhetranscripts by >20°C, which is significantly greater than stabilization by the linear Cdp. In non-denaturing gel electrophoresis, strong and specific binding to Taa, but not to Cdp, was clearly observed for tRNAPhe. In both types of experiments, polyamines and monovalent metal ions competed for binding sites. Structural probing revealed no significant conformational changes in tRNA on Taa binding. In post-transcriptionalin vitromethylation reactions, the formation of m2G/m22G by the methyltransferase Trm1p and of m1A by TrmIp were not affected or only slightly stimulated by polyamines. In contrast, Taa specifically inhibited Trm4p-dependent formation of m5C only in tRNAPhe, likely by occupying sites that are relevant to RNA recognition by the methyltransferase.