Transfer RNA cross-linked with formaldehyde

Abstract

1. 1. The methylene bridges formed during the reaction of formaldehyde with tRNA were situated within the tRNA molecules rather than between them as revealed by sedimentation analysis. The methylene cross-links in tRNA prevented the large tRNA fragments (halves) obtained by limited guanylo-ribonuclease digestion from departing from each other. Hence, the reaction of formaldehyde with tRNA resulted in intramolecular transverse links. 2. 2. Chromatography and analytical ultracentrifugation revealed no damage to the tRNA polymer chain after a long formaldehyde treatment. 3. 3. The native conformation of tRNA was fixed by methylene cross-links only in the presence of Mg 2+; in the absence of the latter, the formaldehyde denatured-conformation was fixed. At 20° and pH 4.8 the reaction of RNA (both highly polymeric and transfer) with formaldehyde was complete within about 15 days. In the absence of Mg 2+, 3–4 methylene bridges were formed per tRNA molecule; in the presence of Mg 2+, one methylene bridge was formed. Prolonged treatment (27 days) did not lead to denaturation of tRNA stabilized with Mg 2+: the amount of CH 2 groups in tRNA did not increase. tRNA treated with formaldehyde in the absence of Mg 2+ after removal of methylol groups by dialysis exhibited no acceptor activity. The same tRNA treated with formaldehyde in the presence of Mg 2+ retained from 23 to 66% of the starting activity depending on acceptor specificity. 4. 4. The behavior of pyrimidyl-ribonuclease digests of cross-linked tRNA in chromatography on DEAE-Sephadex was studied. 5. 5. The reaction of tRNA with bifunctional reagents, in particular with formaldehyde, is proposed as an approach to studying the conformation of tRNA's.