Sequence and secondary structure of the Pyllaiela littoralis (PL) large ribosomal subunit group II intron and the D5 RNA 2D structure as deduced from the NMR data. (A) D5 (circled) is shown in the context of the rest of the “Six-wheel” domain architecture of the PL group II intron. The D5-PL sequence highlighting sites for metal ion binding, intronic component binding, and chemistry are shown together with the extended U6 intramolecular stem–loop structure. The sequences for D5-PL and yeast D5-Ai5γ differ in only three positions indicated by magenta circles. The corresponding Ai5γ sequence of U27–A8 and C25 are indicated with arrows pointing from the D5-PL to D5-Ai5γ sequence. The AGC triad nucleotides are highlighted in magenta for both D5 and U6 ISL. The various tertiary interactions between D5 and D123 are also indicated. (B) NMR evidence for Watson-Crick base pairs in D5-PL shown in A using direct detection of Watson-Crick G–C and U–A base pairs at pH 6.5 and 298 K. Except for U9 and G26, all the imino correlations are observed. (C) Sequential base-ribose walk for the bulge region in D5-PL. A 500 MHz 1H–1H NOESY spectrum of D5-PL was acquired at 45°C in D2O on an ~1 mM RNA sample in 10 mM phosphate buffer (pH 6.5) and 100 mM KCl. The NOESY mixing time was 250 msec. The sequential walk is continuous from G8 to C11 and C23 to A28 and depicted, for clarity, only for the bulge region.
