Structure and stability correlation of an mRNA pseudoknot

Abstract

-1 translational frameshifting is a mechanism employed by the aphid-transmissible plant luteovirus, beet western yellows virus (BWYV), to control the expression of an RNA-based RNA polymerase (P2) from overlapping P1-P2 open reading frames. A heptanucleotide sequence and a downstream pseudoknot are essential for mRNA frameshifting. The BWYV pseudoknot is a classical H-type pseudoknot containing two helical stems and two connecting loop regions. The loop-stem interactions have been proposed to substantially contribute to the net stability of the molecule. Here, we provide thermodynamic and structural evidence for the contribution of the tertiary loop 2-stem 1 interactions towards the global stability of the pseudoknot. BWYV RNAs containing 2' deoxy or A --> G substitutions that disrupt loop 2-stem 1 interactions reduce the net free energy of unfolding to different extents, depending on the location of the mutations with respect to the helical junction. Mutations nearer the helical junction appear to be tolerated by the pseudoknot conformation, while mutations more distal to the helical junction are globally destabilizing. These results suggest that the loop-stem interactions display complementarity and cooperativity that is maximized in the folded structure.