Preparation of partially 2H/13C-labelled RNA for NMR studies. Stereo-specific deuteration of the H5'' in nucleotides

Cromsigt, Jenny; Schleucher, Jürgen; Gustafsson, T.; Kihlberg, Jan; Wijmenga, Sybren S.

doi:10.1093/nar/30.7.1639

Cited by 18 publications

(12 citation statements)

References 28 publications

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“…13 C‐ and 15 N‐labelled NTPs were used to uniformly label the UCAC RNA hairpin. 13 C and 15 N uniformly labeled UCAC sample was prepared from 20 ml transcription reactions using 1 m M NTPs of each 29. The RNA samples were purified on a denaturing 20% polyacrylamide gel and eluted with RNA elution buffer (0.5 M NH 4 OA C , 1 m M EDTA, pH 7.0).…”

Section: Methodsmentioning

confidence: 99%

UNAC tetraloops: to what extent do they mimic GNRA tetraloops?

et al. 2012

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The structures of four small RNAs each containing a different version of the UNAC loop were determined in solution using NMR spectroscopy and restrained molecular dynamics. The UMAC tetraloops (where M is A or C) exhibited a typical GNRA fold including at least one hydrogen bond between the first U and fourth C. In contrast, UGAC and UUAC tetraloops have a different orientation of the first and fourth residues, such that they do not closely mimic the GNRA fold. Although the UMAC tetraloops are excellent structural mimics of the GNRA tetraloop backbone, sequence comparisons typically do not reveal co‐variation between the two loop types. The limited covariation is attributed to differences in the location of potential hydrogen bond donors and acceptors as a result of the replacement of the terminal A of GNRA with C in the UMAC version. Thus, UMAC loops do not readily form the common GNRA tetraloop‐receptor interaction. The loop at positions 863‐866 in E. coli 16S ribosomal RNA appears to be a major exception. However, in this case the GNRA loop does not in fact engage in the usual base to backbone tertiary interactions. In summary, UMAC loops are not just an alternative sequence version of the GNRA loop family, but instead they expand the types of interactions, or lack thereof, that are possible. From a synthetic biology perspective their inclusion in an artificial RNA may allow the establishment of a stable loop structure while minimizing unwanted long range interactions or permitting alternative long‐range interactions. © 2012 Wiley Periodicals, Inc. Biopolymers 97: 617–628, 2012.

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Section: Methodsmentioning

confidence: 99%

UNAC tetraloops: to what extent do they mimic GNRA tetraloops?

et al. 2012

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“…Unlabeled RNA samples were prepared from 20 ml transcription reactions using 4 mM 5′-NTPs as described elsewhere (33–36). Aliquots (1 mM) of 13 C- and 15 N-labeled ATP and GTP (Silantes) were used to uniformly label the RNA samples in 20 ml transcription reactions (37). The RNA samples were purified by electrophoresis on 20% polyacrylamide denaturing gels and eluted with RNA elution buffer (0.5 M NH 4 OA C and 1 mM EDTA, pH 7.0).…”

Section: Methodsmentioning

confidence: 99%

NMR structure and Mg2+ binding of an RNA segment that underlies the L7/L12 stalk in the E.coli 50S ribosomal subunit

Zhao¹

2005

Nucleic Acids Research

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Helix 42 of Domain II of Escherichia coli 23S ribosomal RNA underlies the L7/L12 stalk in the ribosome and may be significant in positioning this feature relative to the rest of the 50S ribosomal subunit. Unlike the Haloarcula marismortui and Deinococcus radiodurans examples, the lower portion of helix 42 in E.coli contains two consecutive G•A oppositions with both adenines on the same side of the stem. Herein, the structure of an analog of positions 1037–1043 and 1112–1118 in the helix 42 region is reported. NMR spectra and structure calculations support a cis Watson–Crick/Watson–Crick (cis W.C.) G•A conformation for the tandem (G•A)2 in the analog and a minimally perturbed helical duplex stem. Mg2+ titration studies imply that the cis W.C. geometry of the tandem (G•A)2 probably allows O6 of G20 and N1 of A4 to coordinate with a Mg2+ ion as indicated by the largest chemical shift changes associated with the imino group of G20 and the H8 of G20 and A4. A cross-strand bridging Mg2+ coordination has also been found in a different sequence context in the crystal structure of H.marismortui 23S rRNA, and therefore it may be a rare but general motif in Mg2+ coordination.

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“…The possibility of incorporating commercially available 13 positions and 13 C labeled on all sugar positions. This approach allowed for the stereo-specific assignment of the H5' resonances, a reduction of spectral crowding and resulted in line narrowing compared with spectra of 13 C labeled non-deuterated RNA [103].…”

Section: Rna Synthesismentioning

confidence: 99%