Thermodynamic analysis of conserved loop-stem interactions in P1-P2 frameshifting RNA pseudoknots from plant Luteoviridae.
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.
Department of Biochemistry and Biophysics, Center for Advanced Biomolecular Research, Texas A&M University, College Station, TX 77843-2128, USA.
Paul L
PL
Nixon
Peter V
PV
Cornish
Saritha V
SV
Suram
David P
DP
Giedroc
D020029:Base Pairing; D001483:Base Sequence; D002151:Calorimetry; D018965:Frameshifting, Ribosomal; D006863:Hydrogen-Ion Concentration; D017793:Luteovirus / Q000235:genetics*; D009682:Magnetic Resonance Spectroscopy; D009690:Nucleic Acid Conformation; D018749:RNA, Plant / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D013816:Thermodynamics
D018749:RNA, Plant