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

β-Galactosidase assay
Overnight cultures were diluted to A 600 of 0.15 and further incubated at 30 o C until
cultures reached A 600 0.3-0.4. Following procedures were performed as described in
(47).
RESULTS
The upstream 34bp fragment of the HSP104 promoter possesses unusual
modular properties
Our previous analysis of the HSP104 promoter revealed that 334bp upstream
of the coding sequence are required for both basal and induced activities whereas
300bp are essential and sufficient for heat shock induced activity only. That is, the
34bp between -334 and -300 are indispensable for the basal activity of the promoter.
Yet, although supporting basal transcription, this 34bp are highly specific to the
HSP104 promoter because they played no role when the upstream fragment of the
HSP104 was fused to the minimal CYC1 promoter. Namely, when cloned upstream
to the CYC1, the fragment between -334 to -160 and the fragment between -305 to -
160 manifested the same, very low basal activity [Fig. 7 and (47)]. These results
suggest that, the activity of the 34bp fragment is not only specific to HSP104, but is
somehow cooperating with the HSP104 minimal promoter (-160-+1) to impose a
relatively high basal activity. Finally, under particular conditions (i.e., in the
ras2∆msn2∆msn4∆ strain) the 34bp acquire new properties and become Ras2
responsive (Fig. 6). Given the importance and the specificity of the 34bp, we
considered that perhaps, there is a shorter cis-element within this sequence. To
address this matter we designed a series of deletion constructs that were planned to
eliminate some potential HSEs present in the 34bp region, as well as a possibly
functional, although non-canonical, TATA box which is also present in this fragment
(Fig. 8A). We also planned a series of constructs that enabled us to monitor the
possible interplay between the 34bp and the minimal promoter. The latter was
achieved by deleting an internal 78bp fragment within the HSP104 promoter which
deletes the two most distal STREs (i.e., at -252 and -220), while leaving intact the
majority of the HSE cluster and the most proximal STRE, residing at -172 of the
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