Identification and topological arrangement of Drosophila proximal sequence element (PSE)-binding protein subunits that contact the PSEs of U1 and U6 small nuclear RNA genes.

In higher eukaryotes, four of the major small nuclear RNAs (snRNAs) found in spliceosomes (U1, U2, U4, and U5) are synthesized by RNA polymerase II (RNAP II), but U6 snRNA is synthesized by RNA polymerase III (RNAP III) (2, 3, 9, 18, 22). The U6 gene promoter is representative of an unusual class of RNAP III promoters that contain a TATA box but lack internal promoter elements (5, 15, 19, 30). Promoters of snRNA genes transcribed by either RNAP II or RNAP III contain an essential proximal sequence element (PSE) at a conserved location approximately 40 to 65 bp upstream of the transcription start site that is required for the initiation of snRNA transcription (4, 9, 18, 26, 29, 30, 32, 36). In Drosophila melanogaster, the PSE is more specifically named the PSEA to distinguish it from a second, even more proximal conserved element, PSEB, present in the promoters of Drosophila snRNA genes transcribed by RNAP II (36). The PSEB is located at the TATA box position but is a poor TATA box sequence (consensus CATGGAg/aA) (16). PSEB is separated from the upstream PSEA by 8 bp of nonconserved sequence (16, 36). Drosophila U6 genes, on the other hand, contain a canonical TATA box rather than a PSEB, and the TATA box is separated by 12 bp from the upstream PSEA (4). In vertebrates and sea urchins, the PSEs of U1 and U2 genes are interchangeable with the PSEs of U6 genes (14, 17, 23). In these organisms, the PSEs are therefore not responsible for the determination of RNAP specificity. Surprisingly, recent results from our lab revealed that the U1 and U6 PSEAs are not interchangeable in the Drosophila system. In fact, the RNAP specificity of Drosophila snRNA genes is determined by the sequence of the 21-bp-long PSEA itself, not by the presence of a TATA box versus PSEB or by differences in the spacing of these elements relative to the PSEA (11). Our results indicated that the U1 and U6 PSEAs, even though they were identical at 16 of 21 base positions, exclusively recruited RNAP II and RNAP III, respectively (11). Further data indicated that all five base differences between the U1 and U6 PSEAs contributed to RNAP specificity, but the differences at base positions 19 and 20 played a major role in determining the RNAP specificity of the Drosophila snRNA promoter (11). The PSE-binding protein (PBP) was first identified in the human system in HeLa cell extracts (31). It was further characterized by two groups and alternatively named proximal transcription factor (PTF) (20) or snRNA activator protein complex (SNAPc) (25). This factor was capable of activating U1 and U2 transcription in vitro by RNAP II and U6 and 7SK transcription by RNAP III (8, 25, 34). PTF and SNAPc each contain four subunits with approximate molecular masses of 180 to 200, 50 to 55, 44 to 45, and 43 to 45 kDa (8, 34). Molecular cloning and sequencing of the three smallest subunits indicated that they are identical in SNAPc and PTF (1, 7, 8, 24, 35). Because each of these subunits is required for snRNA transcription by either RNAP II or RNAP III, it seems that the same protein complex functions at both classes of vertebrate snRNA gene promoters. The fact that the PSEA plays a major role in determining RNA polymerase specificity in Drosophila poses the question of whether a similar complex functions at both RNAP II and RNAP III snRNA promoters in insects. Our lab recently reported the partial purification and characterization of the D. melanogaster PSEA-binding protein (DmPBP) (27). Like SNAPc/PTF, DmPBP is a multisubunit protein, and it is capable of specifically activating transcription of Drosophila U1 and U6 genes in vitro in a soluble nuclear extract (27). By using a site-specific protein-DNA photo-cross-linking technique (13), we here show that DmPBP contains three subunits that are in close proximity to the DNA. The site-specific cross-linking assay has further allowed us to map the translational and rotational positions of these subunits relative to the PSEA. Importantly, the data also reveal that significant conformational differences exist in the DmPBP-DNA complexes depending on whether they are assembled with the U1 or U6 PSEA sequence. These differences may lead to the recruitment of polymerase-specific factors in subsequent steps of preinitiation complex assembly.