Fractionation of HeLa cell extracts reveals the presence of a promoter-specific transcription factor, Sp 1, which activates a class of promoters that includes the SV40 early promoter but not several others that have been tested. We analyzed SV40 early-promoter deletion mutants and determined that transcriptional activation by Sp 1 requires sequences within tandem 21 bp repeats located 70-110 bp upstream of the transcription initiation sites. In a DNAase footprinting assay, Sp 1 protected sequences in this same 21 bp repeat region, thus indicating the presence of a specific site for Sp 1 binding. During purification of Sp 1, there was a correlation between transcription-stimulatory activity and promoter-binding activity. These results suggest that direct binding of Sp 1 to sequences in the upstream promoter element is the mechanism by which this factor activates transcription by RNA polymerase II at the SV40 early promoter.

The enzymatic machinery that carries out RNA synthesis provides the cell with the means to adjust the patterns of transcription in response to environmental and developmental signals. In eukaryotes, this regulation is mediated in part by promoter-specific transcription factors, which are DNA-binding proteins with the ability to discriminate between distinctive DNA sequence elements found in the promoter regions of different genes. The presence of these factors bound to DNA enables other components of the transcriptional machinery, including the RNA polymerase, to initiate transcription with selectivity and accuracy.

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The carboxyl-terminal domain of RNA polymerase U contains a tandemly repeated heptapeptide sequence. Previous work has show that this sequence is phosphorylated at multiple sites by a template-associated protein kinase, in a reaction that is closely associated with the Initiation of RNA synthesis. We have purified this kinase to apparent homogeneity from human (HeLa) cells. (4,5). The CTD is composed of repeats of the heptapeptide (SerPro-Thr-Ser-Pro-Ser-Tyr) (6, 7). Phosphorylation occurs cooperatively at multiple serine and threonine residues and induces a conformational change, as evidenced by altered immunoreactivity and sedimentation rate (8,9). The CTD is essential in yeast, Drosophila, and cultured murine cells (10-13). Mutations in the CTD impair the response of RNAP II to certain transcriptional activator proteins (14)(15)(16) ITo whom reprint requests should be addressed. 11920The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.