FIGURE 1.
Truncation of the polypyrimidine tract (PPT) stalls splicing prior to the first catalytic step. (A) Schematic of PM5 pre-mRNA constructs used, where the polypyrimidine tract was truncated to the indicated length. All constructs have a 6-nt stretch between the branch point adenosine and PPT. Kinetics of in vitro splicing (B,D), and splicing complex formation (C,E) with the PM5 pre-mRNA and truncated versions thereof. Pre-mRNAs were incubated under splicing conditions in the presence of HeLa nuclear extract for the times indicated above each lane. RNA was analyzed by denaturing PAGE and visualized by autoradiography (B,D) The pre-mRNA and splicing intermediates are indicated on the right or left. Spliceosomal complex formation was assayed on a native agarose gel. The positions of the H/E, A, B, C, and Bact complexes are indicated. The arrow indicates the position of the Bact complex. Asterisk, unknown contaminating band.