Enzymatic methylations: III. Cadaverine-induced conformational changes of E.coli tRNAfMet as evidenced by the availability of a specific adenosine and a specific cytidine residue for methylation

Abstract

A partially purified tRNA methylase fraction from rat liver containing m2G- ++m1A- and m5C-methylase, was used to study the influence of Mg++ and of the biogenic polyamine cadaverine on the enzymatic methylation of E.coli tRNAfMet in vitro. In presence of 1 or 10 mM Mg++, guanosine no. 27 was methylated to m2G. In 1 mM Mg++ plus 30 mM cadaverine, guanosine in position 27 and adenosine in position 59 were methylated. In presence of 30 mM cadaverine alone tRNAfMet accepted three methyl groups: in addition to guanosine no. 27 and adenosine no. 59 cytidine no. 49 was methylated. In order to correlate tRNAfMet tertiary structure changes with the methylation patterns, differentiated melting curves of tRNAfMet were measured under the methylation conditions. It was shown that the thermodynamic stability of tRNAfMet tertiary structure is different in presence of Mg++ or Mg++ plus cadaverine, or cadaverine alone. From the differentiated melting curves and from the methylation experiments one can conclude that, at 37° in the presence of Mg++ tRNAfMet has a compact structure with the extra loop and the TφC-loop protected by tertiary structure interactions. In Mg++ plus cadaverine, the TφC-loop is available, while the extra loop is yet engaged in tertiary structure (G-15: C-49) interactions. In cadaverine alone, the TφC-loop and the extra loop are free; hence under these conditions the open tRNAfMet clover leaf may be the substrate for methylation. In general, cadaverine destabilizes tRNAfMet tertiary structure in the presence of Mg++, and stabilizes tRNAfMet tertiary structure in the absence of Mg++. This may be explained by a competition of cadaverine with Mg++ for specific binding sites on the tRNA. On the basis of these experiments a possible role of biogenic polyamines in vivo may be discussed: as essential components of procaryotic and eucaryotic ribosomes they may together with ribosomal factors facilitate tRNA-ribosome binding during protein biosynthesis by opening the tRNA tertiary structure, thus making the tRNA's TφC-loop available for interaction with the complementary sequence of the ribosomal 5S RNA. © 1974 Oxford University Press.