Transcription of a Drosophila U1 small nuclear RNA gene was functionally analyzed in cell extracts derived from 0- to 12-h embryos. Two promoter elements essential for efficient initiation of transcription in vitro by RNA polymerase II were identified. The first, termed PSEA, is located between positions -41 and -61 relative to the transcription start site, is crucial for promoter activity, and is the dominant element for specifying the transcription initiation site. PSEA thus appears to be functionally homologous to the proximal sequence element of vertebrate small nuclear RNA genes. The second element, termed PSEB, is located at positions -25 to -32 and is required for an efficient level of transcription initiation because mutation of PSEB, or alteration of the spacing between PSEA and PSEB, severely reduced transcriptional activity relative to that of the wild-type promoter. Although the PSEB sequence does not have any obvious sequence similarity to a TATA box, conversion of PSEB to the canonical TATA sequence dramatically increased the efficiency of the U1 promoter and simultaneously relieved the requirement for the upstream PSEA. Despite these effects, introduction of the TATA sequence into the U1 promoter had no effect on the choice of start site or on the RNA polymerase II specificity of the promoter. Finally, evidence is presented that the TATA box-binding protein is required for transcription from the wild-type U1 promoter as well as from the TATA-containing U1 promoter.

In a wide variety of eukaryotic organisms, transcription of small nuclear RNA (snRNA) genes is dependent upon a proximal sequence element (PSE) located upstream of position -40 relative to the transcription start site. There is little or no existent knowledge concerning the PSE-binding proteins of organisms other than human. Here, we report the purification of a fraction enriched in the Drosophila melanogaster PSE-binding protein (DmPBP). DmPBP forms a highly specific complex with the PSE. The protein stimulates transcription from the U1 gene promoter by RNA polymerase I1 and from the U6 gene promoter by RNA polymerase I11 in Drosophila nuclear extracts, and activation is dependent upon the presence of a PSE. The molecular mass of native DmPBP as measured by gel-filtration chromatography is 375 kDa. Two polypeptides (apparent molecular masses 59 kDa and 61 kDa) appear to be in close contact with the DNA in that they can be very efficiently and specifically crosslinked to the PSE sequence by ultraviolet irradiation.Keywords: small nuclear RNA gene ; proximal sequence element ; proximal-sequence-element-binding protein; transcription factor; transcriptional activation.The genes encoding the spliceosomal snRNAs can be divided into two classes depending upon whether they are transcribed by RNA polymerase I1 (RNAP 11) or by RNA polymerase I11 (RNAP 111). The U1, U2, U4 and U5 snRNA genes are transcribed by RNAP 11, whereas U6 genes are transcribed by RNAP 111. In vertebrates, transcription of both classes of snRNA genes is dependent upon the proximal sequence element (PSE), a cis-acting element that is located upstream of position -40 with respect to the transcription start site. The PSE is unique to snRNA genes and to a few other related genes that encode small stable cellular RNAs (reviewed by Dahlberg and Lund, 1988;Parry et al., 1989;Hernandez, 1992; Lob0 and Hernandez, 1994).Cis-acting elements that are functionally homologous to the vertebrate PSE have been identified at similar positions upstream of the RNAP 11-transcribed and RNAP 111-transcribed snRNA genes of sea urchins, insects and nematodes (Li et al., 1996;Zamrod et al., 1993;Thomas et al., 1990;Hannon et al., 1994). A functionally similar regulatory element termed the USE is present upstream of plant snRNA genes (Goodall et al., 1991). Based upon these observations, it is evident that the PSE of snRNA genes is an ancient regulatory element that has been conserved functionally (but not in sequence) from early in the evolutionary history of eukaryotic organisms. The human PSE-binding protein has been purified from HeLa cell extracts and partially characterized. Different research groups have termed it the PSE-binding protein (PBP), proximal transcription factor (PTF), or small nuclear RNA activator protein complex (SNAPc) (Waldschmidt et al., 1991;Murphy et al., 1992;Simmen et al., 1992;Sadowski et al., 1993). It is capable of activating U1 and U2 gene transcription by RNAP 11, as well as U6 and 7SK gene transcription by RNAP I11 (Sadowski et al., 1993;...

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Transcription of a Drosophila U1 small nuclear RNA gene was functionally analyzed in cell extracts derived from 0- to 12-h embryos. Two promoter elements essential for efficient initiation of transcription in vitro by RNA polymerase II were identified. The first, termed PSEA, is located between positions -41 and -61 relative to the transcription start site, is crucial for promoter activity, and is the dominant element for specifying the transcription initiation site. PSEA thus appears to be functionally homologous to the proximal sequence element of vertebrate small nuclear RNA genes. The second element, termed PSEB, is located at positions -25 to -32 and is required for an efficient level of transcription initiation because mutation of PSEB, or alteration of the spacing between PSEA and PSEB, severely reduced transcriptional activity relative to that of the wild-type promoter. Although the PSEB sequence does not have any obvious sequence similarity to a TATA box, conversion of PSEB to the canonical TATA sequence dramatically increased the efficiency of the U1 promoter and simultaneously relieved the requirement for the upstream PSEA. Despite these effects, introduction of the TATA sequence into the U1 promoter had no effect on the choice of start site or on the RNA polymerase II specificity of the promoter. Finally, evidence is presented that the TATA box-binding protein is required for transcription from the wild-type U1 promoter as well as from the TATA-containing U1 promoter.