Thermodynamic Analysis of Conserved Loop−Stem Interactions in P1−P2 Frameshifting RNA Pseudoknots from Plant Luteoviridae

Abstract

The RNA genomes of plant luteovirids beet western yellows virus (BWYV), potato leaf roll virus (PLRV), and pea enation mosaic virus (PEMV RNA1; PEMV-1) contain a short mRNA pseudoknotted motif overlapping the P1 and P2 open reading frames required for programmed -1 mRNA ribosomal frameshifting. The relationship between structure, stability, and function is poorly understood in these RNA systems. A m(5)-C(8)-substituted BWYV RNA is employed to establish that the BWYV P1-P2 pseudoknot is protonated at cytidine 8 in loop L1 (delta(N(3)H)+ = 12.98 ppm), which stabilizes a C(+.)(G-C) major groove base triple by Delta(DeltaG(37))(protonation) = 3.1 (+/-0.4) kcal mol(-1). The stabilities of both the PLRV and PEMV-1 P1-P2 pseudoknots are also strongly pH-dependent, with Delta(DeltaG(37))(protonation) = 2.1 (+/-0.2) kcal mol(-1) for the PEMV-1 pseudoknot despite a distinct structural context. As previously found for the BWYV pseudoknot [Nixon and Giedroc (2000) J. Mol. Biol. 296, 659], both the PLRV and PEMV-1 RNAs are stabilized by DeltaH > or = 30 kcal mol(-)(1) in excess of secondary structure predictions, attributed to loop L2-stem S1 minor groove triplex interactions. BWYV RNAs containing single 2'-deoxy or A --> G substitutions that disrupt L2-S1 hydrogen bonding are strongly destabilized with Delta(DeltaG(37))(folding) (pH = 7.0) ranging from approximately 1.8 (+/-0.3) to > or =4.0 kcal mol(-1), relative to the wild-type BWYV RNA. These findings suggest that each member of this family of pseudoknots adopts a tightly folded structure that maximizes the cooperativity and complementarity of L1-S2 and L2-S1 loop-stem interactions required in part to offset the low intrinsic stability of the short three base pair pseudoknot stem S2.

A loop 2 cytidine-stem 1 minor groove interaction as a positive determinant for pseudoknot-stimulated –1 ribosomal frameshifting

“…This destabilization is manifest over the entire pH range investigated (Fig. 7B), with a shift in the macroscopic unfolding pK a for C8 N3 protonation by 0.4 units (23,24). These data reveal that at least part of the global destabilization must be localized to the helical junction given the downward shift in the pK a .…”

“…Our data are consistent with a scenario where a local destabilization allows the elongating ribosome to unfold the distal pseudoknot-forming stem S2 before significant partitioning into the Ϫ1 reading frame. In fact, although the data are limited, the destabilization separating wild-type and C27A ScYLV RNAs is similar in magnitude to the degree to which the helical junction regions are destabilized in other frameshifting systems (⌬⌬G 37 Ϸ 1-2 kcal⅐mol Ϫ1 ) due to substitutions or deletions of junction nucleotides that negatively impact frameshift stimulation (24,28,29,40,48,49). Indeed, if the entire ground-state destabilization of mutant pseudoknots observed here (⌬⌬G 37 Ϸ 1.5 kcal⅐mol Ϫ1 ) is manifest as a decrease in the energy barrier to ribosome-promoted pseudoknot unwinding, ⌬⌬G ‡ , then this would translate into an Ϸ10-fold increase in the rate of unfolding in destabilized pseudoknots (14).…”

“…All RNA constructs were prepared by runoff transcription using SP6 RNA polymerase and purified as described in ref. 24. The final NMR buffer was 10 mM potassium phosphate, 100 mM KCl, and 5 mM MgCl 2 (pH 6.0), with RNA concentrations ranging from Ϸ0.9 to 2.0 mM.…”

“…Hairpin-type luteoviral mRNA pseudoknots from BWYV, pea enation mosaic virus 1 (PEMV-1) and potato leaf roll virus (PLRV) have been previously studied by NMR, x-ray crystallographic, and thermodynamic methods (4)(5)(6)(20)(21)(22)(23)(24)(25). Each of these pseudoknots adopts a unique triple helical architecture that is characterized by an adenosine stack at the 3Ј end of L2, which forms hydrogen bonding interactions into the minor groove of the base of S1.…”

“…Each of these pseudoknots adopts a unique triple helical architecture that is characterized by an adenosine stack at the 3Ј end of L2, which forms hydrogen bonding interactions into the minor groove of the base of S1. These pseudoknots also contain a protonated C ϩ ⅐(G-C) L1-S2 major groove Hoogsteen-type base triple at the helical junction of S1 and S2, with protonation strongly stabilizing these RNAs by 2-3 kcal⅐mol Ϫ1 at 37°C (23,24). The closing base pair of S1 stacks on the L1-S2 C ϩ ⅐G Hoogsteen base pair, creating a strongly over-rotated and horizontally displaced helical junction, allowing the 3Ј adenosine base of L2 to stack directly on S2.…”