Initial analysis of 750 MHz NMR spectra of selectively ¹⁵N‐G,U labelled E. coli 5S rRNA

Abstract

The overall folding of an RNA molecule is reflected in its base pairing pattern. The identification of that pattern provides a first step towards the determination of the structure of an RNA molecule. We show that the application of heteronuclear NMR methods at 750 MHz to E. coli 5S rRNA (120 nucleotides) selectively labelled with 15N in guanine and uridine allows observation of base pairing patterns for a larger RNA molecule. We also present evidence that the fold of the E-domain of the 5S rRNA (nt 79-97) as a contiguous part of the 5s rRNA and as an isolated molecule is virtually the same.

Distinguishing Inter- and Intrastrand NOEs Involving Exchangeable Protons in RNA Duplexes

“…For example, our recent experdecoupled signal in Fig. 4C are roughly 0.4 (G 5 -U 7 ), 0.7 (G 5 -U 6 ), and 1.5 (G 5 intranucleotide); i.e., even in this iments with selectively { 15 N} -G,U-labeled Escherichia coli 5S rRNA (16) showed 1 H linewidths of about 50-60 Hz small palindromic duplex there are some NOE cross peaks for which the undecoupled pulse sequence of Fig. 2B gives for imino protons of G and U bases.…”

Triple-Resonance Experiments for Correlation of H5 and Exchangeable Pyrimidine Base Hydrogens in 13C,15N-Labeled RNA

“…Figures 3B and 3C illustrate the chemical shift correlations achieved with the sequences for the uridines and cytidines in the uniformly 13 C, 15 N-labeled 43-nucleotide RNA shown in Fig. 3A.…”

“…In principle, selective C-N transfers are also possible by a heteronuclear C-N TOCSY mixing scheme (19 -21) as in (4,5), but the use of selective INEPT transfer steps allows the concatenation of different refocusing and defocusing periods, thereby leading to a further reduction of the net transfer time. The heteronuclear TOCSY mixing also requires the application of long, high power RF irradiation [ϳ90 ms, 1.9 kHz in (4)] on the 15 N channel, which Resonances belonging to the same amino group are connected by a dotted line. The spectrum was acquired with 64 ‫ء‬ 1024 complex points in t 1 and t 2 , respectively, spectral widths of 2000 Hz in F1 and 13,000 Hz in F2, 256 scans per t 1 increment, and a relaxation delay of 2 s. The experiments were performed on a 0.8 mM uniformly 13 C, 15 N-labeled RNA sample at 25°C on a Varian Unity INOVA 600 MHz spectrometer.…”

“…3A. The sample was prepared by in vitro transcription with T7 RNA polymerase (13) and a linearized plasmid template using uniformly 13 C, 15 N-labeled nucleotides essentially as described previously (14 -16). The NMR sample contained 0.8 mM RNA, 60 mM KCl, 5 mM potassium phosphate buffer, and 6 mM CaCl 2 at pH 6.0 in a volume of 600 l. The lengths of the transfer delays and ⌬ were optimized experimentally for this molecule to ϭ 3.4 and ⌬ ϭ 10 ms.…”

“…At point c in the sequence the 15 N magnetization is antiphase with respect to the C4 carbon (N3 X C4 Z / N4 X C4 Z ). During the next interval 2⌬ this magnetization is refocused with respect to the C4 -N3/C4 -N4 coupling and allowed to evolve due to the N3-H3/N4 -H4 coupling so as to lead to the formation of N3 X H3 Z /N4 X H4 Z at point d. To take into account the different number of 15 N attached hydrogens in the case of uridines and cytidines, the time for the evolution due to the N-H scalar coupling is set to different durations in the two cases. In the case of the uridine optimized sequence, a selective 180°pulse on C4 is used to avoid leakage of magnetization through the unwanted N3-C2 pathway.…”