Nuclear magnetic resonance investigation of the base-pairing structure of Escherichia coli rRNATyr monomer and dimer conformations

Abstract

The structures of the E. coli tyrosine tRNA monomer and dimer were investigated by high-resolution NMR. At 23.degree. C the monomer contains 26 .+-. 2 base pairs and the low-field NMR spectrum (11.7-15 ppm) can be accounted for in terms of the cloverleaf structure (23 base pairs) and 3 additional resonances that are assigned to tertiary structure base pairs. Assignments suggested for the various resonances are consistent with thermal denaturation studies in low-salt solutions. Under these conditions the temperature dependence of the spectrum can be interpreted in terms of sequential unfolding of the cloverleaf structure with the minor and dihydrouridine stems melting first, followed by the T.psi.C stem, the anticodon stem, and finally the amino acceptor stem. Certain features of the tertiary structure of tRNATyr are similar to other tRNA, but some details of the folding must be different, since no resonance from the s4U8.cntdot.A14 tertiary base pair is observed. The tRNATyr dimer contains only 20 .+-. 2 base pairs per tRNA (40/dimer) at 23.degree. C and a good account of the low-field NMR spectrum can be given in terms of a secondary structure in which bases of the T.psi.C stem and loop are involved in intermolecular base pairing. Formation of the dimer requires opening of the hU and T.psi.C stems, but not the anticodon or amino acid acceptor stems, and this fits well with relative stabilities observed for these stems in the monomer. The model also provides an explanation for the formation of 2n-mers, that were stable enough to be separated by gel electrophoresis at room temperature (10 mM Mg2+). Experimental conditions required for interconversion of monomer and dimer are described.