Effect of a quaternary pentamine on RNA stabilization and enzymatic methylation.
Extreme 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 methylated in vitro by purified tRNA methyltransferases under various salt conditions. At concentrations in the micromolar range, branched Taa causes a considerable increase in the melting temperature (T(m)) of yeast tRNA(Phe) transcripts by >20 degrees 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 tRNA(Phe). 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-transcriptional in vitro methylation reactions, the formation of m(2)G/m(2)(2)G by the methyltransferase Trm1p and of m(1)A by TrmIp were not affected or only 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.
Hayrapetyan
Armine
A
Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Heidelberg, Germany.
Grosjean
Henri
H
Helm
Mark
M
D004586:Electrophoresis; D008745:Methylation / Q000187:drug effects*; D009690:Nucleic Acid Conformation / Q000187:drug effects; D011073:Polyamines / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology*; D020871:RNA Stability / Q000187:drug effects*; D044366:Transition Temperature / Q000187:drug effects; D014466:Ultraviolet Rays; D012359:tRNA Methyltransferases / Q000378:metabolism
D011073:Polyamines; D000644:Quaternary Ammonium Compounds; C035585:caldopentamine; C040648:pentamine; D012359:tRNA Methyltransferases