Nuclear Overhauser assignment of the imino protons of the acceptor helix and the ribothymidine helix in the nuclear magnetic resonance spectrum of Escherichia coli isoleucine transfer ribonucleic acid: evidence for costacked helices in solution.

In a previous study we showed that, in the low-field nuclear magnetic resonance (NMR) spectrum of Escherichia coli tRNAVal1, the hydrogen-bonded imino protons of the four Watson-Crick base pairs in the dihydrouridine helix could be assigned on the basis of their proximity to the imino proton of s4U8 by means of sequential Nuclear Overhauser (NOE) connectivity (Hare & Reid, 1982). In the present paper we have used the nearest-neighbor NOE technique to assign all the imino proton resonances of the acceptor helix and the ribothymidine helix of E. coli tRNAIle1. As reference points we used the GU-type base pairs located at positions 5 and 49 in this molecule which are readily identifiable in the NMR spectrum by virtue of containing two imino protons in the same base pair. From UG5 the imino protons of base pairs 4,3,2,1 and 6,7 could be assigned by through-space NOE connectivity. Similarly the imino protons of 50,51,52,53 were assigned by their spatial relationship to G psi 49. NOE connectivity also revealed a base pair stacked on the external side of GC53 which, by analogy with the crystal structure of yeast phenylalanine tRNA, is presumed to be the tertiary pair T54-A58. This was confirmed by NOE connectivity from the thymine methyl resonance. In addition to assigning 17 of the imino resonances in the low-field NMR spectrum of isoleucine tRNA, these NOE studies show that the acceptor helix and the ribothymidine helix are stacked on each other in solution in that base pairs 7 and 49 are directly connected in space.