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

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Using PCR on genomic DNA we cloned a fragment of 713bp of the promoter and ligated it upstream to a β-galactosidase reporter gene (Fig. 2A). When introduced to yeast cells, the -713LacZ reporter gene manifested basal activity under non heat shock conditions which was induced 5.5 fold in response to heat shock (Fig. 2B). This reporter activity reflected the levels of endogenous HSP104 mRNA that accumulated thirty minutes after heat shock and dropped after one hour (Fig. 2C). We next proceeded with 5’-deletions in order to determine the minimal promoter sequence conferring basal and induced activities. This deletion analysis (Fig. 2A) showed that a fragment of 334bp upstream from the first AUG gave rise to reporter activities similar to the full length -713LacZ. A drastic decrease in basal (but not induced) reporter activity was observed upon further removal of 34bp [-300LacZ (Fig. 2B)]. These results strongly suggested that 334bp of the promoter are essential and sufficient for the basal activity of HSP104 promoter. 300bp of the promoter are essential and sufficient for heat shock-induced activity. Namely, the 34bp between - 334 and -300 are dispensable for induced activity, but indispensable for HSP104 transcription under optimal growth conditions. Further analysis of those 34bp is described below. 11
A) 5’-XhoI -713 BamHI-3’ ATG LacZ coding seq -641 ATG -531 ATG -500 ATG -334 ATG -300 ATG Legend HSE HSE cluster STRE-like STRE 100 bp B) C) 600 500 400 SP1 30 0 C 39 0 C Time in 39 0 C 0’ 15’ 30’ 60’ 5hrs 30 0 C SP1 HSP104 ACTIN 300 200 100 0 -713 -641 -530 -500 -334 -300 Figure 2. A 334bp fragment of the HSP104 promoter is sufficient and essential for both basal and induced activities in wild-type cells (the SP1 strain). A) Schematic view of various constructs fused to LacZ coding sequence, ranging from -713bp to -300bp of the promoter. B) β-galactosidase activity of the various constructs under optimal growth conditions (30 o C) and following heat shock (39 o C for one hour). C) S1 analysis of endogenous HSP104 mRNA at various time points during heat shock treatment or under optimal growth conditions. In order to search for the cis-elements required for the heat shock induced transcription of HSP104, the sequences downstream to -300 were further analyzed through 5’deletions. The results are described in detail in (47). Briefly, we found that upon deletion of the HSE cluster (Fig. 3) between -300 and -286, the reporter gene remained responsive to heat shock due to the presence of the STREs of the promoter (reflected by the -284LacZ construct). Removal of the first distal STRE positioned at -252 (in the -248LacZ construct) almost abolished the responsiveness of the reporter gene. Only residual activity remained that was just slightly induced in response to heat shock. This induced activity of -248LacZ is due to the presence of the remaining STREs positioned at -220 and -172 because their deletion completely abolished reporter activity (Fig. 3B and 3C). As mentioned above, the general stress response via the STRE/Msn2/4 system is negatively regulated by the Ras/cAMP/PKA pathway and many stress related genes are upregulated in ras2∆ cells (9, 86, 116, 121, 129). 12
Page 1 and 2: Revealing the Mechanism of HSP104 T
Page 3 and 4: This thesis is dedicated to my fami
Page 5 and 6: genetic approach, we identified com
Page 7 and 8: INTRODUCTION The cellular stress re
Page 9 and 10: for their transcription, some 16% o
Page 11 and 12: Transcription under stress in S.cer
Page 13 and 14: In mammalian cells, Hsf1 is a monom
Page 15: Hsf1 and Msn2/4 can exclusively or
Page 19 and 20: 1 We first noticed that the activit
Page 21 and 22: 1 1 A) B) 700 600 500 ras2∆msn2
Page 23 and 24: Table 1. Summary of the role of var
Page 25 and 26: same enzymes. BS-HA-HSF was obtaine
Page 27 and 28: β-Galactosidase assay Overnight cu
Page 29 and 30: to the fact that the HSEs are prese
Page 31 and 32: The systematic 5’ deletion analys
Page 33 and 34: 1 A) units 100.00 90.00 80.00 70.00
Page 35 and 36: nucleosome disassembly involving sp
Page 37 and 38: A) Strain W.T. msn2∆msn4∆ ras2
Page 39 and 40: Hsf1 constitutively binds the HSP10
Page 41 and 42: Next, we attempted to directly meas
Page 43 and 44: Table 3. List of mutants in which r
Page 45 and 46: Table 4. Strains required for posit
Page 47 and 48: Overall, the results nevertheless s
Page 49 and 50: Regulation of Hsf1 As explained in
Page 51 and 52: Table 7. Comparison between HSE-Lac
Page 53 and 54: promoter. III) The identification o
Page 55 and 56: to stress. This change in nucleosom
Page 57 and 58: perhaps not surprisingly, overexpre
Page 59 and 60: 21. Chandy, M., J. L. Gutierrez, P.
Page 61 and 62: 56. Hietakangas, V., J. K. Ahlskog,
Page 63 and 64: 89. Miyao, T., J. D. Barnett, and N
Page 65 and 66: 121. Stanhill, A., N. Schick, and D

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