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Figure 2.
Figure 2. Elongation Defects and Altered Substrate Selection
by Rpb1 H1085Y Pol II
(A) H1085Y exhibits reduced
elongation rate using NTP substrates. Run-off transcription of
an oligonucleotide scaffold template generates a 61 nt RNA
product. Representative experiments for WT and H1085Y Pol II are
shown in the left and right panels, respectively. Average
elongation rates for each NTP concentration were measured as the
length of the transcribed region (51 nt) divided by the time of
half-maximal accumulation of run-off product (61 nt). Average
elongation rates were then plotted versus NTP concentration to
infer maximum average elongation rate (see Experimental
Procedures for details) (top right graph). Inferred maximum
average elongation rates are shown in the bottom right graph
with error bars representing the 95% confidence interval (See
Experimental Procedures for details).
(B) H1085Y Pol II
exhibits only modest defects for 2′-dNTP incorporation. WT and
H1085Y Pol II ECs were formed on oligonucleotide scaffolds
containing 10-mer RNAs with templates specifying addition of
different NTPs at position 11. Average incorporation rates for
different template-specified 2′-dNTPs were measured as
1/t[1/2] for maximal accumulation of 11-mer RNA. Incorporation
rates were then plotted versus 2′-dNTP concentration, and
maximum incorporation rate for either 2′-dATP or 2′-dGTP was
inferred (left panels). Maximum incorporation rates for WT Pol
II and H1085Y are shown in the right panels with error bars
representing the 95% confidence interval (See Experimental
Procedures for details).
(C) H1085Y Pol II exhibits modest
defects in GTP misincorporation. WT and H1085Y ECs were formed
and labeled as in (B) with templates specifying incorporation of
ATP at the position being measured, but were challenged with 1
mM GTP and misincorporation rate measured as the 1/t[1/2] for
maximal incorporation. Mean misincorporation rate from at least
three experiments is represented in the bar graph (error bars
represent ± SD).
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