Sp1 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 degrees 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 Sp1. Gel mobility shifts indicated that Sp1 DNA binding activity was heat-labile, and the addition of purified Sp1 to 40 degrees C heat-treated extracts fully restored DHFR transcriptional activity. In contrast, the addition of Sp1 to 47 degrees C heat-treated extract did not result in transcriptional activity from the DHFR promoter. We conclude that reduction in Sp1 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 degrees C heat-treatment. The discovery of this heat-labile component of Sp1 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 Sp1-activated promoters. Second, it explains the previously reported low-temperature optima for transcription from the DHFR promoter and demonstrates that transcriptional studies of Sp1-activated promoters should not be performed at 30 degrees C.
Farnham
P J
PJ
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Cornwell
M M
MM
D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000502:physiology*; D006367:HeLa Cells; D006358:Hot Temperature; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000502:physiology*; D016329:Sp1 Transcription Factor / Q000502:physiology*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000502:physiology; D014158:Transcription, Genetic / Q000502:physiology*
D004268:DNA-Binding Proteins; D016329:Sp1 Transcription Factor; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D013762:Tetrahydrofolate Dehydrogenase; D012319:RNA Polymerase II