HIV-1 frameshift efficiency is primarily determined by the stability of base pairs positioned at the mRNA entrance channel of the ribosome

Mouzakis, Kathryn D.; Lang, Andrew L.; Vander Meulen, Kirk A.; Easterday, Preston D.; Butcher, Samuel E.

It has been hypothesized that during frameshifting, the mechanical force of translocation causes a build-up of tension that is transmitted through the spacer region ( Figure 1A ) and sensed at the anticodon-codon level (5, 49) . We therefore investigated the influence of nt deletions and insertion in the spacer region ( Figure 4 ). The WT construct was compared to a version with an adenosine insertion that increases the spacer length by 1 nt (MS13) (Figure 4) . Additionally, we created spacers with a single nt deletion (MS14) and 2-nt deletions (MS15-17) (Figure 4 ). The resulting frameshift efficiencies were measured ( Figure 4D ). Interestingly, MS15 shows a large increase in frameshift efficiency. This cannot be due to the 2-nt deletion, since MS16 and MS17 have the same spacers yet display WT-levels of frameshifting. We hypothesized that the 2-nt deletion in the spacer of MS15 increased frameshift efficiency by altering the base pairs in the stem-loop encountered by the ribosome during frameshifting. In other words, by deleting 2 nt in the spacer, a ribosome footprint may extend 2 nt further into the stem. In support of this hypothesis, a very stable set of base pairs are located 2 nt from the base of the stem (5 0 -GGC-3 0 /5 0 -GCC-3 0 ). In MS16 and MS17, we replaced these base pairs with the less stable base pairs normally found at the base of the stem (5 0 -CUG-3 0 /5 0 -CAG-3 0 ) ( Figure 4C ). Indeed, when these changes are made, the frameshifting efficiency is indistinguishable from WT, despite the apparent 2-nt spacer deletion ( Figure 4D) . Interestingly, the overall stability of MS17 is increased relative to MS16 ( Figure 4C ), yet the frameshift efficiency is unaltered ( Figure 4D ). These data indicate that changes in the spacer region correspondingly alter the base pairs encountered by the ribosome when it is engaged with the slippery site.