IDENTIFICATION OF 4-THIOURIDINE IN ESCHERICHIA COLI tRNMer AND tRNAiyr

Uridine is uniquely conserved at position B in elongator tRN As and binds to A 14 to form a reversed Hoogsteen base pair which folds the dihydrouridine loop back into the core of the L-shaped molecule. On the basis of IH NMR studies, Hurd and co-workers (Hurd, R. E., Robillard, G. T., and Reid, B. R. (1977) Biochemistry 16,2095-2100) concluded that the interaction between positions Band 14 is absent in Escherichia coli tRNAs with only 3 base pairs in the dihydrouridine stem. We have taken advantage of the unique 1liN chemical shift of N3 in thiouridine to identify IH and 1liN resonances for the imino units of s4UB and s"U9 in E. coli tRNAt'r and tRNA~Yr. Model studies with chloroform-soluble derivatives of uridine and 4thiouridine show that the chemical shifts of the protons in the imino moieties move downfield from 7.9 to 14.4 ppm and from 9.1 to 15.7 ppm, respectively; whereas, the corresponding 1liN chemical shifts move downfield from 157.5 to 162.5 ppm and from 175.5 to 1BO.1 ppm upon hydrogen bonding to 5'-O-acetyl-2',3'-isopropylidene adenosine. The large difference in 1liN chemical shifts for U and s"U allows one to unambiguously identify s"U imino resonances by 1liN NMR spectroscopy. E. coli tRNAt'r and tRNA~yr were selectively enriched with 1liN at N3 of all uridines and modified uridines. Two-dimensionaI1H-1IiN chemical shift correlation NMR spectroscopy revealed that both tRN As have resonances with IH and 1liN chemical shifts characteristic of s"UA pairs. The IH shift is approximately 1 ppm upfield from the typical s"UB resonance at 14.B ppm, presumably as a result of local diamagnetic anisotropies. An additionals U resonance with IH and 1liN • hifts typical of interaction of a bound water or a sugar hydroxyl group with sU9 was discovered in the spectrum of tRNA~yr_ Our NMR results for tRNAs with 3base pair dihydrouridine stems suggest that these molecules have an UBA14 tertiary interaction similar to that found in tRN As with 4-base pair dihydrouridine stems.