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. 2015 Dec 15;44(4):1845–1853. doi: 10.1093/nar/gkv1381

Figure 7.

Figure 7.

Ll.LtrB circularization pathway. Ll.LtrB-WT (WT) and Ll.LtrB-ΔA (ΔA) use the same circularization pathway (A–C) in vivo. (A) The first nucleophilic attack at the intron-exon 2 junction is initiated by the 3′-OH of a free exon 1 (step 1). Accurate recognition of the 3′ splice site properly ligates the flanking exons leading to the expression of an active relaxase (Active LtrB). The 2′-OH of the last intron residue initiates the second nucleophilic reaction at the exon 1-intron junction (step 2) resulting in precise intron circularization and the release of free exon 1 available to initiate another circularization reaction. (B and C) The 3′-OH of a free exon 1 misrecognizes the intron-exon 2 junction during the first nucleophilic attack (step 1) leaving tails of 2 (CA) (B) or 3 (CAU) (C) nts at the 3′ end of the intron. Misrecognition of the 3′ splice site also improperly ligates the flanking exons creating a premature stop codon just after the splice junction (-CA) (Inactive LtrB, LtrB-ΔE2) (B) or deleting an important catalytic amino acid from the LtrB relaxase enzyme (-CAU) (Inactive LtrB, LtrB-ΔHis) (C). The 2′-OH of the first nt of exon 2 then accurately initiates the second nucleophilic reaction at the exon 1-intron junction (step 2) resulting in its incorporation at the intron circularization junction and the release of free exon 1 available to initiate another circularization reaction. Ll.LtrB-WT circularization predominantly involves the release of perfect intron circles (A) while the great majority of Ll.LtrB-ΔA circles harbor the first nt of exon 2 at their splice junction (B, C). However, low level of accurate (perfect intron circles, precisely ligated exons, SF conjugation) and inaccurate (first nt of exon 2 at circle splice junction) circularization can still be detected for Ll.LtrB-ΔA and Ll.LtrB-WT respectively. Removal of the branch point A residue significantly affects 3′ splice site recognition tilting the balance from accurate (A) to inaccurate (B and C) 3′ splice site recognition ultimately leading to the addition of the first nt of exon 2 (C) at the circle splice junction (cf. WT (open arrows) and ΔA (black arrows)).