The 22/6a and the 0 constructs are the two constructs with the lowest frequency of frameshifting. Therefore, they have the least ribosome traffic into the lacZ sequence. Alteration of the UAA stop codon into a lysine AAA codon in the spacer between the slippery sequence and the pseudoknot changed the pattern of transcripts immensely. These two downstream stop variants ('DS. stop' in Figure 6 ), which did not contain a stop codon upstream from the structure, expressed significantly more full-length ('Fl') transcript and only insignificant amounts of premature transcription stop fragment ('SP') compared to their sister constructs containing the UAA stop codon upstream from the structure ( Figure 6 ). Our control construct, PK401, which stimulated 14% frameshift, showed no premature transcription stop fragment ('SP') and therefore no change in transcription pattern was observed as a consequence of removing the upstream UAA stop codon ( Figure 6 ) thus confirming that the major effect causing the 'SP' fragment is polarity in the lacZ gene and not transcription termination caused by the pseudoknot sequences. Also, the very short band marked by 'asterisks' that appeared from the 22/6a construct was not present in the 'DS. stop' variant ( Figure 6 ). This exclude this mRNA fragment to be causal for the appearance of stalled protein products, because 22/6a ('DS. stop') is the construct that caused the highest frequency of stalling (compare Figure 2 and Supplementary Figure S5 ).