Revealing the Mechanism of HSP104 Transcription Initiation in the ...

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1
A) B)
700
600
500
ras2∆msn2∆msn4∆
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14
12
30 0 C
Time in 39 0 C 0’ 15’ 30’ 60’
Sp1ras2∆msn2∆msn4∆
5hrs
30 0 C
HSP104
ACTIN
400
300
10
8
6
200
4
100
25.5
2
0
-334 -300 -284 -280 -260
0
-248 -230 -222 -215 -200 -180
Figure 6. HSP104 expression in ras2∆msn2∆msn4∆ cells. A) Sequences between 334 and 300bp
of HSP104 are important for the β-galactosidase activity of the promoter in ras2∆msn2∆msn4∆ cells at
30 o C. A change in scale used in the right hand graph. The 25.5 fold decrease in activity was obtained
by dividing the activity of -334LacZ by that of -300LacZ. B) S1 analysis of HSP104.
In order to unambiguously assess that the sequences identified in our study are
indeed independently responsible for the heat shock responsiveness of the HSP104
promoter, we fused the sequences from -334 to -160, or -305 to -160 (these sequences
contain all elements responsible for the induced activity, but lack the basal promoter
region) to the CYC1 minimal promoter and checked whether the sequences derived
from the HSP104 promoter could now render the CYC1 promoter heat shock
responsive [the native CYC1 promoter is normally not induced by heat shock (data not
shown)]. Briefly, we found that the HSP104 enhancer region is indeed sufficient for
rendering the heterologous promoter responsive to heat shock (Fig. 7A). Also, the
heterologous reporters were constitutively elevated in ras2∆ cells. Namely, the
sequence we defined as an enhancer is indeed, independently, necessary and sufficient
for promoter activation and regulation in response to heat shock and in response to the
Ras pathway. Notably however, we also observed that the heterologous promoters
displayed lower basal activity compared to their homologous counterparts (compare
Fig. 7A and 2B and data not shown). These results suggest that the element we
defined as essential for the basal transcription activity (i.e., the 34bp between -334
and -300) are specific for the HSP104 promoter and functions together with its own
basal promoter and cannot function with another.
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