The results shown in Figure 2 revealed that a 1D SDS-PAGE assay could not firmly identify polypeptides originating from a À1 frameshifted ribosome stalled in the pseudoknot from the non-frameshifted product in a 'Downstream Stop' construct. In order to quantify the amount of À1 frameshifted ribosomes stalled inside the pseudoknot, we performed a 2D SDS-PAGE separation of the radioactively labelled proteins originating from the 'Upstream Stop' construct (Supplementary Figures S4 and  S5) , which is the type of construct most commonly used throughout literature. The advantage of a 2D-gel analysis is that all the unfinished protein chains with different lengths concentrate in a common spot when they have the same pI. This made it possible to identify randomly stalled translation products inside the pseudoknot sequence and we quantified the amount of radioactivity in all identified additional spots. This produced a conservative estimate of the amount of stalled translations.