The 1.7 Angstrom X-ray crystal structure of an RNA pseudoknot involved in translational frameshifting from Beet Western Yellow Virus has been solved. The RNA pseudoknot tertiary folding motif is topologically formed through the hairpin loop structure. In this structure, a continuous single stranded region outside of the hairpin folds back to pair with bases in its loop region. Three dimensionally, the structure consists of a quasi-continuous helix Stem 1 and Stem 2, with Loop 1 crossing the major groove of Stem 2 and Loop 2 crossing the minor groove of Stem 1. This unique structure can perturb the translational machinery, resulting in a -1 frameshifting at the upstream slippage site, a stop codon readthrough, to produce a single fusion protein. The crystal structure of this 28 nucleotide frameshifting pseudoknot reveals significant differences from the regular A-form RNA helix and non-coaxial characteristics at the two stems. At the junction of the two stems, the disruption of A25-U13 base pairing serves as an anchor to bend the quasi-continuous helical conformation. In addition, base C8 in Loop 1 forms base triplet interactions with the major groove residues while residue A9 stacks on the end of Stem 2. With the exception of G19, the longer Loop 2 stacks continuously in the minor groove and forms several triple base and 2 OH interactions with bases in Stem 1. At the junction of Stem 1 and Loop 2 there is a sharp turn that is facilitated by C2 endo sugar pucker conformations. These features along with the continuous stacking of Loop 2 into Stem 2 contributes to the overall non-coaxial characteristics of the molecule. This crystal structure of a pseudoknot is near completion in refinement with a current R factor of 20.4% and R free of 25.2%. In general, the structural analysis implies that loop sequence and length can influence the overall configuration of the pseudoknot, primarily at the hinge between the two quasi-continuous stems. The structure will reveal in some detail how magnesium ions stabilize this motif. Furthermore, it may also provide insight on how proteins and the translational machinery interact with this specific pseudoknot conformation to allow frameshifting, which is an essential feature in many viral systems.
Showing the most recent 10 out of 375 publications
