New insights into the transposition mechanisms of IS6110 and its dynamic distribution between Mycobacterium tuberculosis Complex lineages
Fig 3
Post-transcriptional regulatory mechanisms of IS6110 translation.
(a) Genetic organization of IS6110. Overlapping ORF1 and ORF2 and the sense of transcription are indicated as blue and red arrows respectively. The scheme also shows the 28bp inverted repeats (IR) flanking both overlapping ORFs. (b) Mechanisms of post-transcriptional regulation of IS6110. The image shows an enlarged view of the region indicated with a dotted box from panel (a). The UUUUAAAG slippery sequence is indicated by a grey box. ORF1 and ORF2 as well as their coding triplets are indicated by blue and red letters according to panel (a). The RNA pseudoknot is included within a red rectangle and those regions involved in base pairing formation of secondary structures are indicated by blue, green and orange boxes. The position of the ribosome and the translated codons are also indicated. Asterisks in the pseudoknot indicate positions carrying mutations that disrupt this structure. (c) Expression of 3xFLAG variants of IS6110-WT, the transcriptionally active transposase IS6110-FS and the latter variant carrying mutations to disrupt pseudoknot formation IS6110-FS+PK. The upper and lower parts of the panel show a western-blot using and anti-FLAG antibody and a Coomassie staining which serves as loading control respectively. The right side of the panel shows the band intensity average from three independent experiments. (d) Post-transcriptional regulation of IS6110 to produce a biologically active transposase. The image shows translation steps indicating the sense of ribosomal advance and the mRNA structure indicated in panel (b). Translation of the ORF1 produces the aminoacids from the N-terminus of IS6110 (blue spheres) until it translates Ile91 and Leu92 coded by AUU and UUA triplets in the slippery region (grey box). At this position ribosome stalls probably because the presence of the downstream pseudoknot presenting a tight secondary structure. Stalling favours a -1 frameshift in the slippery region. Translation continues in the AAA codon coding for the Lys1 position of ORF2 (red sphere) until the ribosome reaches the C-terminus of IS6110 coded in this latter ORF.