Structure of transfer RNA by carbon NMR: resolution of single carbon resonances from¹³C-enriched, purified species

Abstract

Methyl carbon-13 NMR spectra of purified tRNA species are presented for the first time. In addition, these spectra of tRNA species specific for phenylalanine, tyrosine, and cysteine exhibited the first resolution of single methyl carbon resonances. Carbon-13 enriched methyl groups of ribothymidine (T) and 7-methylguanosine (m7G) and the methylthio group of 2-methylthio-N6-(delta2-isopentenyl) adenosine (ms2i6A) were resolved. The T methyl signal of tRNAPhe shifted from 12.3 ppm at 45 degrees in the absence of added Mg2+ to 11.1 ppm at 30 degrees in the presence of 10mM MgCl2. The same change in conditions led to a 0.4 ppm shift of the m7G methyl signal in the opposite direction. The relative ease in obtainment of single carbon resonances of purified tRNA species, and display of the sensitivity of their chemical shifts to changes in local structure, are requisite criteria for 13C-NMR to be a useful technique in probing tRNA conformation and its changes during interaction with proteins and other nucleic acids.

13C NMR studies of methylnucleosides

“…13C NMR has also been used in studying the biochemical modification of RNA. 12,13 This direct method of analysis does not require tedious degradation or separation. Therefore, it eliminates artifacts resulting from secondary chemical alteration and/or decomposition.…”

Chemical modification of ribonucleic acid. A direct study by carbon-13 nuclear magnetic resonance spectroscopy

“…This approach also eliminates the interference of 13C natural abundance signal and thus enhances the specificity. Other laboratories have taken an alternative approach to improve the sensitivity and specificity (Agris & Schmidt, 1980;Agris et al, 1975;Schweizer et al, 1980) by using a nutritional auxotroph to specifically incorporate 13C-enriched precursors into RNA. In principle, 2H NMR in conjunction with a high-field spectrometer may provide better specificity to detect the low degree of the reactions, such as an in vivo system, than 13C NMR since the natural abundance of deuterium (0.015%) is much lower.…”