Next, we investigated the role of stem length on frameshifting efficiency. Increasing stem size from 12 to 15 or 21 bp did not significantly alter frameshifting (Figure 2A ). On the other hand, decreasing stem size led to a steady decrease in frameshifting efficiency which seemed to vanish around a stem size of 5 bp or ÁG 37 of À7.7 kcal/ mol ( Figure 2B ). Thermodynamic stabilities were calculated at the MFOLD website using version 2.3 parameters (http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form2.3), as these were previously shown to better fit in vivo hairpin stabilities (29) . These data support the notion that downstream structures serve as barriers to stall translating ribosomes to stimulate frameshifting, and demonstrate that there is a correlation between the thermodynamic stability of a hairpin and its frameshift inducing capacity.