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Direct Evidence for Watson-Crick Base Pairs in a Dynamic Region of RNA Structure



B. Luy, John Marino


Trans-H-bond 2hJN1N3 scalar couplings were measured for the RNA hairpin, CopA29, which is derived from the R1 plasmid encoded RNA transcript, CopA,21 using a dual-detected uracil H3/adenine-H2 (UH3/AH2) Jnn HNN-COSY (see supporting Information for details), which is an adapted version of the previously described JNN-HNN-COSY.9 The experiment simultaneously correlates both the exchangeable uracil H3 proton to uracil N3 H-bond donor and adenosine N1 H-bond acceptor and uracil N3 H-bond donor nitrogens in AU base pairs that are 2h JNIN3 Scalar coupled (Figure1). Cop A29 has a predicted secondary structure (Figure 2A) that is comprised of a lower and upper helical stem region separated by a single bulged G nucleotide and a six-nucleotide loop. In disagreement with the predicted secondary structure, AU base pair associated imino proton resonances have been observed for only the lower stem base pairs in this RNA using conventional one-and two-simensional NMR techniques. Figure 2B shows selected regions of the JNN-COSY experiment applied to a uniformly 15N-labeled 29 nucleotide CopA29 hairpin at 25 C. In the CopA29 RNA hairpin, five H-bonded AU base pairs are expected based on the predicted secondary structure. Inspection of the adenosine H2 -> uracil N3 cross-peak region of the UH3/ AH2 JNN HNN-COSY experiment clearly reveals seven, rather than the expected five, correlations. Four H2, N3 correlations are assigned to the lower stem AU base pairs (U4-A27, U5-A26 and U6-A25) and three H2, N3 correlations are assigned to the upper stem base pairs (U21-!10, U12-A19). Note that the additional correlations observed for base pairs U21-A10 and U6-A25 arise from two different conformational states of the bulge that are in slow-exchange on the NMR time scale. In contrast, the H3 detected regions of the experiment show only the four H3, N1 correlations associated with the AU base pairs in the lower stem. Thus, these measurements directly demonstrate AU base pairing in a region of RNA strlucture that is involved in intermediate conformational exchange.
Journal of the American Chemical Society
No. 33


heteronuclear nuclear magnetic resonance, hydrogen bond, J-couplings, RNA


Luy, B. and Marino, J. (2000), Direct Evidence for Watson-Crick Base Pairs in a Dynamic Region of RNA Structure, Journal of the American Chemical Society (Accessed April 19, 2024)
Created August 22, 2000, Updated October 12, 2021