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

1
We first noticed that the activity of the full length -334LacZ was decreased
when compared to wild type, indicating some possible role for Msn2/4 in the basal
activity of HSP104 (compare Fig. 5 with Fig. 3). However, unexpectedly, the reporter
gene was induced to maximal levels even in the absence of these two transcriptional
activators suggesting that the induced activity could be solely provided by the
HSE/Hsf1 system. Indeed, when we deleted the HSE cluster (reflected by the -
284LacZ construct), and subjected msn2∆msn4∆ cells to heat shock, the reporter gene
was no longer induced in response to heat shock, confirming that in msn2∆msn4∆
cells, heat shock responsiveness of the reporter gene is due to the HSEs alone (Fig. 5).
This conclusion is further strengthened by measurements of the mRNA levels of
HSP104 in msn2∆msn4∆ cells which are normally induced in response to heat shock.
A) B)
900.00
800.00
700.00
600.00
500.00
400.00
ras2∆
40.00
35.00
6.5
30.00
25.00
20.00
-HS
Time in 39 0 C 0’ 15’ 30’ 60’
SP1ras2∆
SP1RAS2 val19
5hrs
30 0 C
HSP104
ACTIN
HSP104
ACTIN
300.00
200.00
100.00
3.3
15.00
10.00
5.00
6
3
1 0.00
-334 -284 -260
0.00
-300 -280 -248 -230 -222 -215 -200 -180
-160
Figure 4. HSP104 expression is derepressed in ras2∆ cells and is regulated exclusively through
STREs. A) Deletion of each STRE causes a decrease in basal (30 o C) β-galactosidase activity of the
promoter (compare 260 vs. 248; 222 vs. 215; and 180 vs. 160). The graphs shown are different in
scale. The numbers above some bars describe the fold reduction in activity as compared with the
previous bar. B) S1 analysis of HSP104 mRNA in ras2∆ and RAS2 val19 cells.
Our deletion analysis, in combination with the effects of the point mutations,
strongly suggests that the derepression of the HSP104 promoter in ras2∆ cells is
mediated exclusively via STREs. These STREs must be recognized by Msn2 and
Msn4, as they are not functional in msn2∆msn4∆ cells (see Fig. 5).
14