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13C-filtered NOESY and TOCSY [G. Otting and K. Wüthrich, Q. Rev. Biophys. 23, 39 (1990); W. Lee et al., FEBS Lett. 350, 87 (1994)]. Sequential connectivities between nucleotides in RNA were obtained by HP-COSY and HCP. Adenine H2 protons were assigned by correlation to H8 resonances by 2D HCCH-TOCSY [J. P. Marino et al., J. Am. Chem. Soc. 116, 2205 (1994)]. The hydrogen bonding patterns of the base pairs were determined from analysis of SSNOESY spectra in water at different mixing times (75, 150, and 300 ms) [S. H. Smallcombe, J. Am. Chem Soc. 115, 4776 (1993)]. For the U-U base pair a strong NOE was observed between the two imino protons.
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10544219953
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note
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Distance restraints for nonexchangeable RNA protons were derived from visual inspection of NOESY cross-peak intensities at 50, 100, 150, 200, and 250 ms NOESY. NOEs were classified into three distance bound ranges: strong 1.8 to 2.5 E̊, medium 2.5 to 3.5 Å. and weak 3.5 to 5 Å (or 3.5 to 5.5 Å for some NOEs with exchangeable protons of the RNA, obtained from 75- and 150-ms SSNOESY experiments, or 3.5 to 6 Å for some intermolecutar NOEs involving dynamic rings of the paromomycin). Appropriate pseudoatom distance corrections were used.
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10544231390
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note
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1495(H3)-ring II(6).
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25
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0029082529
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H1′-H2′ was >8 Hz or <3 Hz, respectively. No restraints were used for riboses with mixed sugar conformations. Several paromomycin ring I and II dihedral angles were restrained from analysis of the short mixing time TOCSY and DQF-COSY spectrum.
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J. Mol. Biol.
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Allain, F.H.-T.1
Varani, G.2
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0028008137
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H1′-H2′ was >8 Hz or <3 Hz, respectively. No restraints were used for riboses with mixed sugar conformations. Several paromomycin ring I and II dihedral angles were restrained from analysis of the short mixing time TOCSY and DQF-COSY spectrum.
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J. Am. Chem. Soc.
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Marino, J.P.1
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10544236051
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note
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1. All color figures were generated with the program Insightll (Biosym Technologies. San Diego, CA).
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10544232542
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note
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We thank H. Noller for encouragement and discussions, A. Pardi for assistance with NMR techniques, E. V. Puglisi for comments and support, and M. Ares and R. Green for reading the manuscript. Supported by grants from the Packard Foundation, the Deafness Research Foundation, National Institute of Health (GM51266-01A1), Lucille P, Markey Charitable Trust (J.D.P.), and a postdoctoral grant from Institut National de la Santé et de la Recherche Médicale (D.F.), The coordinates have been deposited in the Brookhaven Protein Data Bank with access number T9496.
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