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Figure 1.
Figure 1 (A) Time course of limited proteolysis. A 42  g
aliquot of CBC was incubated at room temperature with 210 ng of
trypsin in the absence or presence of 10 mM cap analogue
m^7GpppG in a total volume of 60 l.
Aliquots of 10 l
were taken off every 0, 5, 10, 20, 40 and 60 min, denatured by 3
l
of denaturing buffer (125 mM Tris pH 6.8, 260 mM DTT, 30%
glycerol, 10% SDS and 0.025% Coomassie Blue) and loaded onto a
13.5% Tricine SDS−polyacrylamide gel. (B) Cap-binding activity
of CBP80  653−701.
[^35S]Methionine-labelled wild-type or mutant CBP80 was
incubated for 30 min at room temperature in the absence (−) or
presence of either 1.7 M
(high) or 53 nM (low) m^7GpppG-capped (m7) or ApppG-capped (A)
unlabelled U1 Sm
RNAs. The samples were fractionated by native 6% PAGE followed
by fluorography. Free CBP80, CBC and the CBC−RNA complex are
indicated. In the lanes indicated by an asterisk, the
corresponding CBP80 proteins were loaded without CBP20. (C)
Shuttling activity of CBP80 NLS
653−701
in Xenopus oocytes. [^35S]methionine-labelled CBP80 NLS2
or CBP80 NLS2
653−701
were injected together with [^35S]methionine-labelled GST−M10
into Xenopus oocyte nuclei either (1) alone (lanes 1 and 2, and
7 and 8), (2) together with m7GpppG-capped unlabelled U1 Sm
RNAs (lanes 3 and 4, and 9 and 10) or (3) together with
ApppG-capped U1 Sm
RNAs (lanes 5 and 6, and 11 and12). Oocytes were dissected
either immediately (lanes 1 and 2, and 7 and 8) or 5 h after
injection (lanes 3−6 and 9−12) and the proteins analysed by
SDS−PAGE followed by fluorography. GST−M10 is a mutant of
HIV Rev with a non-functional nuclear export signal used as a
negative control. See Ohno et al. (2000) or Segref et al. (2001)
for more details.
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