We also checked whether the protein products from within the pseudoknot structure could arise from very slow rather than permanently stalled ribosomes. A pulse chase experiment (Supplementary Figure S2 ) revealed that within 16 min there was no sign of a redistribution of label between the stalled spots and the stop codon-terminated downstream stop product, thus proving the possibility of very slow ribosomes to be unlikely. It is possible that the newly discovered ribosome rescue factor, ArfA (39) could be active at pseudoknot-stalled ribosomes and that nascent proteins would be more stable than if saved by tmRNA. However, as can be seen in Supplementary Figure S3 , the growth of strains expressing pseudoknot 22/6a was severely affected by induction and showed a decrease in growth rate correlating to the amount of stall product observed. Because ribosomes are limiting in growing cells (40) , the sequestration of ribosomes by engagement in induced overexpression of a gene from a plasmid will often cause a strain to grow slower than the uninduced counterpart. The enhanced reduction in growth rate upon induction of 22/6a compared to the 0 construct (Supplementary Figure S3 ) could indicate that stalled ribosomes were not rescued at a sufficiently high rate and we suggest that either the ribosomal rescue systems were titrated by the large amount of mRNA induced from the plasmid alleles, or alternatively, that no rescue is possible for pseudoknot-stalled ribosomes.