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.