High-resolution nuclear magnetic resonance determination of transfer RNA tertiary base pairs in solution. 2. Species containing a large variable loop

Abstract

The number of base pairs in the solution structure of several class III D3VN tRNA species from E. coli has been determined by analyzing the number of low-field (-15 to -11 ppm) proton resonances in their nuclear magnetic resonance spectra at 360 MHz. Contrary to previous reports indicating the absence of tertiary resonances, all the spectra exhibit the expected number of secondary base pair resonances plus approximately ten extra resonances derived from tertiary base pairs in the three-dimensional folding of these molecules. The possible origins of some of these tertiary resonances are discussed; none of the spectra exhibits the characteristic resonance of the 8-14 tertiary base pair seen in class I D4V5 tRNA spectra.

Structure of an Escherichia coli tRNA operon containing linked genes for arginine, histidine, leucine, and proline tRNAs

“…It is not known whether this material corresponds to a genetically unique species or a variant of a normal species. Some have suggested it is simply a nuclease-nicked form of tRNAIeu (18), but this material could also be a modification-deficient form known to elute in this region (27,28). Both possibilities are consistent with the observation that the presence of this species is variable and often undetected (Fournier et al, unpublished data).…”

“…For the quantitation of leucine tRNA synthetase activities, protein concentrations were determined by the procedure of Bradford (3), and enzyme activity was measured in reactions which were 100 mM sodium cacody-late (pH 8.2), 60 mM MgC92, 4 mM ATP, 4 mM NH4CI, 2.5 mM ,-mercaptoethanol, and 15 ,uM [3H]leucine and contained 1 absorbance unit at 260 nm (A260) of unfractionated tRNA and a limiting amount of enzyme as determined in preliminary assays. Acylation assays for histidine and proline were carried out under similar conditions, except the reaction mixtures were 50 mM Tris-hydrochloride (pH 7.5), 18 mM MgCl2, 4 mM ATP, 10 mM ,-mercaptoethanol, 5 mM NH4Cl, and radioactive amino acid at 10 to 50 ,uM. All reactions were done at 37°C.…”

Effect of cations on tRNA structure

“…In presenting the results we will use the spectrum of tRNA in solutions containing 0.17 M Na-10 mM Mg at pH 7 (referred to as native tRNA) as a reference. Native class I tRNAs exhibit 24 ± 2 resonances in the low-field region (Bolton et al, 1976;Bolton andKearns, 1975, 1976; Cohn, 1975, 1976;Geerdes and Hilbers, 1977;Robillard et al, 1976;Reid et al, 1977;Hurd et al, 1977). This indicates that there are several tertiary interactions which have resonances in this region in addition to the resonances from secondary structure base pairs.…”

Complete nuclear magnetic resonance signal assignments and initial structural studies of [13C]methyl-enriched transfer ribonucleic acid

“…the approximately 20 hydrogen-bonded base pairs contributing to the secondary structure (Kim et al, 1974). Proton NMR studies of tRNA have exclusively dealt with these hydrogen-bonded protons (Reid & Robillard, 1975;Hurd et al, 1977; Reid et al, 1977;Rómer & Varadi, 1977) with the exception of some recent studies of methyl protons (Koehler & Schmidt, 1973; Kan & Ts'o, 1974; Kastrup & Schmidt, 1975;Kan et al, 1977; Schmidt & Kastrup, 1978). Hydrogen-bonded protons produce signals that are relatively easy to assign in contrast to signals from non-hydrogen-bonded protons and are of interest because of their importance to the basic structure of the molecule.…”