Spl activation of RNA polymerase II transcription complexes involves a heat-labile DNA-binding component
We have identified a component of the eukaryotic RNA polymerase II transcriptional machinery that is more heat-labile than TFIID. DHFR transcriptional activity was severely reduced in 40°C heat-treated extracts in which TFIID was fully active. This heat-labile activity was required for the transcription of both TATA box and non-TATA box promoters that are activated by the transcription factor Spl. Gel mobility shifts indicated that Spl DNA binding activity was heat-labile, and the addition of purified Spl to 40°C heat-treated extracts fully restored DHFR transcriptional activity. In contrast, the addition of Spl to 47°C heat-treated extract did not result in transcriptional activity from the DHFR promoter. We conclude that reduction in Spl DNA binding activity is partially responsible for the heat-sensitive loss of DHFR transcriptional activity, but that a second essential activity is also inactivated by 47°C heat-treatment. The discovery of this heat-labile component of Spl activation has two important implications in the analysis of transcriptional regulation. First, it demonstrates that heat-treated extracts are not appropriate for examination of the involvement of TFIID in the transcription of Spl-activated promoters. Second, it explains the previously reported low-temperature optima for transcription from the DHFR promoter and demonstrates that transcriptional studies of Spl-activated promoters should not be performed at 30°C.
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Document Type: Research Article
Publication date: January 1, 1991
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