An examination of coaxial stacking of helical stems in a pseudoknot motif:The gene 32 messenger RNA pseudoknot of bacteriophage T2

The RNA pseudoknot located at the 5' end of the gene 32 messenger RNA of ba cteriophage T2 contains two A-form helical stems connected by two loops, in an H-type pseudoknot topology. A combination of multidimensional NMR metho ds and isotope labeling were used to investigate the pseudoknot structure, resulting in a more detailed structural model than provided by earlier homo nuclear NMR studies. Of particular significance, the interlace between the stacked helical stems within the pseudoknot motif is described in detail. T he two stems are stacked in a coaxial manner, with an approximately 18 degr ees rotation of stem1 relative to stem2 about an axis that is parallel to t he helical axis. This rotation serves to relieve what would otherwise be a relatively close phosphate-phosphate contact at the junction of the two ste ms, while preserving the stabilizing effects of base stacking. The ability of the NMR data to determine pseudoknot bending was critically assessed. Th e data were found to be a modestly precise indicator of pseudoknot bending, with the angle between the helical axes of stem1 and stem:! being in the r ange of 15 +/- 15 degrees. Pseudoknot models with bend angles within this r ange are equally consistent with the data, since they differ by only small amounts in the relatively short-range interproton distances from which the structure was derived. The gene 32 messenger RNA pseudoknot was compared wi th other RNA structures with coaxial or near-coaxial stacked helical stems.