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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 4. Positional flexibility and preferred identity of the branch nucleophile in orthogonal BS-U2 systems.

Figure 4

(A) Schematic of reporter and U2 constructs used in (B).

(B) Primer extension on total RNA from the indicated strains with 3′ exon (panel i), BS-proximal intronic (panel ii), and 5′SS-proximal intronic (panel iii) primers indicates that two bulge positions in this duplex are catalytically competent, that the expected branch nucleotide is used in each case, and that the location of the branch nucleotide in the BS-U2 duplex does not affect the position of 5′SS cleavage. Panel iv, quantitation of the 3′ exon primer extension in (i).

(C) Schematic of reporter and U2 constructs used in (D).

(D) Primer extension on total RNA from the indicated strains primers as in (B) indicates that three bulge positions in this duplex are catalytically competent, and, as in (B), that the branch nucleophile can be moved without changing the position of 5′SS cleavage.

(E) Schematic of reporter and U2 constructs used in (F).

(F) Primer extension using a 3′ exon primer indicates an intrinsic nucleotide preference at the branch site, with A>G>>C~U.

(G) Schematic of reporter and U2 constructs used in (H).

(H) Primer extension indicates that branching occurs from the upstream-most nucleotide in a run of consecutive single-stranded adenosines.