C O M M U N I C A T I O N S
Table 1. Transcriptional Activation of HIS3 and lacZ in Yeast L40
This hypothesis is supported by the observation that the transcrip-
tional activation of lacZ in this system increases with the concentra-
tion of 2 and 3, whereas a quantitative labeling would result in a
decrease of transcriptional activation of lacZ with increasing
concentration of CID.9 At 1, 10, and 100 µM of 3, the measured
ONPG hydrolysis in cell extracts was 39, 830, and 8100 nM min-1
mg-1, respectively (Supporting Information). For 2, the measured
â-galactosidase activity in cell extracts was 13, 865, and 2600 nM
min-1 mg-1 at concentrations of 0.1, 1, and 10 µM of 2, respectively
(Supporting Information). The relatively low solubility of 2 made
measurements at higher concentrations impossible.
To evaluate the achieved transcriptional activation using CID 2,
we measured the transcriptional activation of lacZ by coexpression
of LexA-Fos and B42-Jun fusion proteins, a protein pair known
to yield a strong interaction signal in the two-hybrid system.10
â-Galactosidase activity in cell extracts of L40 expressing LexA-
Fos and B42-Jun was below the activity measured for LexA-
3HYAGT and B42-DHFR in the presence of 1 µM CID 2 (Table
1). Furthermore, the observed 200-fold transcriptional activation
at 1 µM of 2 over background is comparable to those achieved
with other noncovalent CIDs in yeast.6,9
growth
ratea
ONPG
hydrolysisb
protein pair
CID
LexA-3HYAGT/
B42-DHFR
1 µM 2
++
870 ( 410
1 µM 3
+
-
-
-
+
39 ( 19
4 ( 2
-
100 µM BG
100 µM Mtx
1 µM 2, 100 µM BG,
100 µM Mtx
1 µM 2, 100 µM BG
1 µM 2, 100 µM Mtx
1 µM 2
4 ( 4
6 ( 4
19 ( 2
+
++
-
41 ( 16
820 ( 210
1 ( 0.3
LexA-A145AGT/
B42-DHFR
LexA-Fos/ B42-Jun
-
++
260 ( 130
a ++: detection of colonies within 3 days; +: detection of colonies
within 6 days; -: no colonies within 7 days. b nM of o-nitrophenol formed
per min per mg total protein. Each value represents the average of at least
three independent experiments.
Coexpression of LexA-3HYAGT and B42-DHFR allowed yeast
L40 to grow on plates lacking histidine but containing either 2 or
3, indicating transcription of HIS3. No growth was observed in the
absence of 2 or 3 or in the presence of only BG or Mtx. For yeast
L40 expressing LexA-3HYAGT and B42-DHFR, 2 proved to be
more effective as a CID than 3, most likely due to differences in
cell permeability of the two CIDs. Yeast L40 coexpressing LexA-
A145AGT and B42-DHFR did not grow on plates lacking histidine
and supplemented with 2, indicating that the growth of the yeast
depends on the labeling of the hAGT fusion protein. For CID 2,
we also investigated if the growth of yeast expressing LexA-
3HYAGT and B42-DHFR on plates lacking histidine but containing
2 could be suppressed by the addition of free Mtx and BG (Table
1). Although the growth rate was significantly lower in the
simultaneous presence of excess Mtx and BG, growth was not
completely suppressed. Addition of only Mtx did not significantly
affect the growth rate. The competition experiments indicate that
the intracellular concentration of the small molecules, and in
particular that of Mtx, is below that of the fusion proteins.
We then examined the activation of transcription of lacZ by the
BG-based CIDs in yeast L40 expressing LexA-3HYAGT and B42-
DHFR fusion proteins (Table 1). In this assay, the activity of the
product of the lacZ gene, â-galactosidase, was determined by
measuring the hydrolysis rate of the chromogenic substrate o-ni-
trophenyl-â-D-galactopyranoside (ONPG) in cell extracts of liquid
cultures.8 The data obtained for the transcriptional activation of
lacZ confirm those observed for HIS3 described above. At 1 µM
CID, 2 leads to about 20-fold higher levels of â-galactosidase
activity compared to 3, and no significant activity was measured
in the absence of CID or when CID was replaced by BG or Mtx.
No transcriptional activation in the presence of 2 was observed
when 3HYAGT was replaced by A145hAGT. The addition of free
Mtx and BG to the culture medium did not lead to complete
suppression of â-galactosidase activity. As observed in the HIS3
assay, adding only Mtx to the medium did not significantly affect
the activity of CID 2, whereas the addition of only BG leads to a
reduction of â-galactosidase activity by a factor of 20. Again, the
lack of (complete) suppression of transcription of lacZ indicates
that the covalent and noncovalent labeling of the 3HYAGT and
DHFR fusion proteins under these conditions is not quantitative.
The work presented here demonstrates that BG derivatives can
be used as CIDs to control transcription in yeast. The specificity
of the labeling of hAGT fusion proteins and its independence of
the nature of the ligand should make the approach a valuable tool
to control protein dimerization in vivo.
Acknowledgment. This work was financed by the Swiss
National Science Foundation, the Bundesamt fuer Bildung und
Wissenschaft (Project X-TB), and the Boehringer Ingelheim
Foundation. We thank Nils Johnsson for valuable advice.
Supporting Information Available: Detailed procedures for
synthesis of 2 and 3 and biochemical assays (PDF). This material is
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