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. Author manuscript; available in PMC: 2010 May 15.
Published in final edited form as: Mol Cell. 2009 May 15;34(3):333–343. doi: 10.1016/j.molcel.2009.03.012

Figure 6. The preferred position of the BS nucleophile within the BS-U2 duplex is partially determined by distance from the U2 component of U2/U6 helix Ia.

Figure 6

For each panel: (i) Simplified schematic of the spliceosome catalytic centre with helices Ia and III indicated; BS adenosines in blue are sized proportionally to their ability to participate in splicing catalysis based on quantitation (v) of the 3′ exon primer extension in (ii). Branch site and 5′SS use are assayed by primer extension in (iii) and (iv), respectively: the position of the branch nucleotide both within the BS-U2 duplex and relative to the U2 component of helix Ia does not impact the position of 5′SS cleavage. All panels use an all-CG BS-U2 duplex, indicated below the figure, in which purine-pyrimidine identity is maintained at each position relative to wild-type.

(A) Profile of −3 to +1 bulged reporters with CG-substituted but otherwise wt U2.

(B) Deletion of two nucleotides in U2 downstream of helix Ia, and insertion of two nucleotides upstream of helix III, predicted to move the BS-U2 duplex towards helix Ia and away from helix III, activates upstream bulge positions for catalysis.

(C) Insertion of one nucleotide in U2 downstream of helix Ia, and deletion of one nucleotide upstream of helix III, predicted to move the BS-U2 duplex away from helix Ia and towards helix III, represses the use of upstream bulge positions for catalysis.

(D) Deletion of two nucleotides in U2 downstream of helix Ia recapitulates the effect seen in (B), activating upstream bulge positions for catalysis.

(E) Deletion of one nucleotide in U2 upstream of helix III does not strongly impact the catalytic potential of different bulge positions.