Transfer RNA contains sites of localized positive charge: carbon NMR studies of [13C]methyl-enriched Escherichia coli and yeast tRNAPhe.

Abstract

The possibility of positively charged nucleosides in tRNA has been suspected because certain posttranscriptional methylations produce quaternary nitrogens. To investigate this possibility and the importance of such methylations to tRNA structure, we have continued our studies of [13C]methyl-enriched phenylalanine tRNA of Escherichia coli [Kopper, R.A., Schmidt, P.G., & Agris, P.F. (1983) Biochemistry 22, 1307-1401] and yeast [Smith, C., Petsch, J., Schmidt, P.G., & Agris, P.F. (1985) Biochemistry 24, 1434-1440]. E. coli and yeast tRNA were 13C-enriched in their methyl groups in vivo, and phenylalanine-specific tRNA was isolated. Methyl proton and carbon signal assignments were confirmed and correlated for the purified tRNAs under native conditions via the first application of two-dimensional carbon-proton correlation NMR spectroscopy to a native nucleic acid. The methyl proton chemical shift of the 7-methylguanosine (m7G) signal from tRNA was easily determined, although by conventional 1H NMR spectroscopy it would have been hidden by ribose resonances and H2O. The chemical shift for 1-methyladenosine (m1A) protons was shown to be 3.01 ppm. Resolution of close or overlapping peaks was greatly enhanced by the two-dimensional experiment especially for the proton methyl resonances. In addition, proton-carbon chemical shift correspondence has been determined for the two 5-methylcytidines (m5C's), the methyl esters of wybutosine (Y), and the two ribose methyl groups, Gm and Cm, of yeast tRNAPhe. Thermal denaturation and Mg2+ depletion affect the methyl carbon NMR chemical shifts of tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)