α 32p gtp

For reactions in Figures 3B and
S2A, 8
μl reactions containing 4 nM of the lacCONS DNA
template, 20 nM RNAP holoenzyme, 100 μM ApA, 1X RB, and 100 nM GreB
(where indicated) were incubated for 2 min at 25°C. For reactions
halted at position +7 (Figure 3B,
left), 2 uL of 50 μM UTP and 50 μM GTP (final concentration of
10 μM UTP, and 10 μM GTP [cold GTP + 5 μCi
32P-α-GTP (Perkin Elmer; 3000 Ci/mmol)]) were added. For reactions
halted at position +11, 2 uL of 50 μM ATP, 50 μM UTP and 50
μM GTP (final concentration of 10 μM ATP, 10 μM UTP,
and 10 μM GTP [cold GTP + 5 μCi 32P-α-GTP (Perkin
Elmer; 3000 Ci/mmol)]) were added. After addition of NTPs, reactions were
incubated for 5 min at 25°C, and 10 μl of 2 X RNA loading dye
was added. Samples were heated at 95°C for 1 min, cooled to room
temperature, and run on 20% TBE-Urea polyacrylamide gels (UreaGel system,
National Diagnostics). Autoradiography of gels was performed as above. The
intensity of the 6- and 11-nt RNA products were used to calculate the
percentage of the total RNA products that were 6 nt using the formula: %
6-nt RNA = 100 x ((6-nt RNA/ (6-nt RNA + 11-nt RNA)). Values reported in
Figures 3B, right and S2B are means
± SD of three independent experiments.

CE-GMP complex formation in the context of permeabilized virions was assayed by incubating varying amounts of purified WT or Dts36 virions in 16 ul reaction mixtures containing 50 mM Tris-HCl (pH 8.2), 10 mM DTT, 0.05% Nonidet P-40, 5 mM MgCl₂ and 1 uCi α³²P-GTP (3000 Ci/mmol, Perkin Elmer). Reactions were incubated either at 30°C or 39°C for 15 min. Products were analyzed by SDS-PAGE and visualized by autoradiography (Shuman & Moss, 1990 (link)). Thermolability of CE-GMP formation in permeabilized virions was analyzed in a similar fashion except virions were pre-incubated at 4°C or 39°C for various lengths of time in reaction buffer lacking GTP. Following pre-incubation, GTP and α³²P-GTP were added to the reactions and reactions were incubated at 39°C for 10 min.
CE-GMP complex formation using purified CE was assayed by titrating purified WT or Dts36 CE in 15 ul reaction mixtures containing 50 mM Tris-HCl (pH 8), 2 mM DTT, 10 mM MgCl₂, 100 uM unlabeled GTP and 3 uCi α³²P-GTP (3000 Ci/mmol, Perkin Elmer). Reactions were incubated at 30°C or 37°C for 15 min, analyzed by SDS-PAGE, and visualized by autoradiography (Shuman & Moss, 1990 (link)). Thermolability of CE-GMP formation by purified CE was analyzed as above except WT or Dts36 CE was pre-incubated at 4°C or 37°C for 15 min.