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

promoter. III) The identification of components of the basal transcription machinery
involved specifically in transcriptionally activating HSP104.
The results obtained allow us to draw a detailed working model for the
molecular events occurring on the HSP104 promoter in response to heat shock (see
details in the last section of the Discussion). The first aspect of our promoter analysis
dealt with, as mentioned above, analyzing the promoter through 5’deletion [most
aspects of promoter analysis are thoroughly described in (47)]. We sought at this
stage of our study, to identify sequences which are required for the high basal activity
of -334LacZ reporter. We found the putative single HSE site between (-304 and -300)
to have an important role in this promoter activity. In fact, our fine-tuned mapping
effort did not point at an additional particular sequence within the 34bp as responsible
for the activity. We further conclude that the 34bp (particularly the HSE between -
304 and -300) may possibly be required to interact with the specific basal
transcription machinery of HSP104 (this conclusion is also based on the results with
the heterologous promoter, Fig. 7). We therefore attempted to analyze the interaction
between the 34bp with the basal transcription machinery by deleting an internal
fragment of 78bp, thereby bringing the 34bp close to the minimal promoter. The
various ∆78 constructs manifested low basal activity, suggesting that perhaps the
spatial organization between the 34bp and the basal transcription machinery is critical
for the basal activity of the HSP104 promoter. It could also be that STREs, removed
with the internal 78bp, are required for basal activity. The ∆78 constructs provided
important information about the flexibility and modularity of the promoter (Fig. 8).
Normally, there is complete cooperation between STRE/Msn2/4 and HSE/Hsf1
systems for optimal promoter activity. Yet, under specific conditions, such as in
msn2∆msn4∆ cells, HSP104 is solely activated by the HSE/Hsf1 system. Conversely,
in ras2∆ cells, the HSE/Hsf1 system is not required for the activation of HSP104
promoter activity. High activity of HSP104 in this strain is due to the hypothesized
constitutive binding of Msn2/4 to the STREs present on the promoter. This
flexibility and back-up capabilities of the HSE/Hsf1 and STRE/Msn2/4 systems are
now further expanded and include the upstream 34bp that become Ras2 responsive in
cells lacking Msn2/4 and the internal 78bp, that under various conditions, affect not
only induced, but also basal promoter activity. Namely, promoter flexibility is much
stronger than we previously suggested and many cis and trans-elements are backing
up each other to allow promoter activity under various circumstances. Measuring
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