Sup. Figure 1 The majority of scaffolds remain stably associated with the template
during competition. a, Imm. 559 Temp. = immobilized 559 template. 515 comp. =
immobilized 515 template competitor. The 559 template is identical to the 515 template
except that it contains a 50 bp deletion in the transcribed region. b, In lanes 1-5 the
stated immobilized templates were incubated with Srb2 NE either in the absence or
presence of rSrb2 for 40 min to form PICs. NTPs were then added for 2 min to allow a
single round of transcription to occur. Reactions performed in lanes 6-14 used scaffolds
formed on the 559 immobilized template as described in a. Scaffolds were incubated
with Srb2 NE in either the absence or presence of 515 competitor for various times. As
a control for residual active complexes no NE was added in lane 6. NTPs were then
added for 2 min to allow a single round of transcription to occur. Reactions were
assayed by primer extension. Brackets indicate the transcription signals. The results
show that after 10 min of competition, most of the transcription originates from the
scaffold template. In Fig. 2, transcription was assayed immediately upon addition of the
second extract, making that signal equivalent to 0 min of competition in Sup. Fig. 1.
Sup. Figure 2 Transcription of a scaffold template occurs at a higher initial rate than
that of a naked template. a, The pSH559 plasmid template was incubated with wildtype NE to form PICs. ATP was added to allow PICs to dissociate into scaffolds. NTPs
were then added for various times either with or without the pSH515 naked plasmid
template. Reactions were assayed by primer extension. The pSH559 plasmid template
is identical to the pSH515 plasmid template except that it contains a 50 bp deletion in
the transcribed region. b, The results of the experiment described in a were plotted as
transcription units vs. time. The primer extension signals were quantitated using the
Phosphorimager, and then normalized to each other.