Transcription of snRNA genes by either RNA polymerase II (U1 to U5) or RNA polymerase III (U6) is dependent upon a proximal sequence element (PSE) located approximately 40 to 60 bp upstream of the transcription start site. In Drosophila melanogaster, RNA polymerase specificity is determined by as few as three nucleotide differences within the otherwise well-conserved 21-bp PSE. Previous photo-cross-linking studies revealed that the D. melanogaster PSE-binding protein, DmPBP, contains three subunits (DmPBP45, DmPBP49, and DmPBP95) that associate with the DNA to form complexes that are conformationally distinct depending upon whether the protein is bound to a U1 or a U6 PSE. We have identified and cloned the genes that code for these subunits of DmPBP by virtue of their similarity to three of the five subunits of SNAP c , the human PBP. When expressed in S2 cells, each of the three cloned gene products is incorporated into a protein complex that functionally binds to a PSE. We also find that the conformational difference referred to above is particularly pronounced for DmPBP45, herein identified as the ortholog of human SNAP43. DmPBP45 cross-linked strongly to DNA for two turns of the DNA helix downstream of the U1 PSE, but it cross-linked strongly for only a half turn of the helix downstream of a U6 PSE. These substantial differences in the cross-linking pattern are consistent with those of a model in which conformational differences in DmPBP-DNA complexes lead to selective RNA polymerase recruitment to U1 and U6 promoters.In eukaryotes, small nuclear RNAs (snRNAs) are required for pre-mRNA splicing. Most snRNAs, such as U1, U2, U4, and U5, are synthesized by RNA polymerase II, but U6 snRNA is synthesized by RNA polymerase III (2,3,6,10,11,18,26). Transcription of snRNA genes by either RNA polymerase is dependent upon a proximal sequence element (PSE) located upstream of position Ϫ40 relative to the transcription start site. In the insect Drosophila melanogaster, the PSE is referred to more specifically as the PSEA to distinguish it from a second conserved element termed the PSEB (38).Although the PSEAs of all D. melanogaster snRNA genes share extensive sequence similarity, the PSEAs of the three U6 genes present in the fly genome consistently vary at a few nucleotide positions from the PSEAs of the RNA polymerase II-transcribed snRNA genes (13). Indeed, RNA polymerase specificity can be determined by as few as three base pair differences within the otherwise well-conserved U1 and U6 PSEAs (13). As a result, the fly U1 and U6 PSEAs are not interchangeable either in vitro or in vivo (13,22).Nonetheless, both PSEAs are recognized by the same D. melanogaster PSE-binding protein, DmPBP (29,33). DmPBP contains three distinct subunits (DmPBP45, DmPBP49, and DmPBP95) that can be specifically photo-cross-linked to DNA containing U1 or U6 PSEA sequences. Interestingly, the pattern of photo-cross-linking was different depending upon whether DmPBP was bound to a U1 or a U6 PSEA (33). Those results, together with the functio...
Transcription of genes coding for metazoan spliceosomal snRNAs by RNA polymerase II (U1, U2, U4, U5) or RNA polymerase III (U6) is dependent upon a unique, positionally conserved regulatory element referred to as the proximal sequence element (PSE). Previous studies in the organism Drosophila melanogaster indicated that as few as three nucleotide differences in the sequences of the U1 and U6 PSEs can play a decisive role in recruiting the different RNA polymerases to transcribe the U1 and U6 snRNA genes in vitro. Those studies utilized constructs that contained only the minimal promoter elements of the U1 and U6 genes in an artificial context. To overcome the limitations of those earlier studies, we have now performed experiments that demonstrate that the Drosophila U1 and U6 PSEs have functionally distinct properties even in the environment of the natural U1 and U6 gene 5-flanking DNAs. Moreover, assays in cells and in transgenic flies indicate that expression of genes from promoters that contain the "incorrect" PSE is suppressed in vivo. The Drosophila U6 PSE is incapable of recruiting RNA polymerase II to initiate transcription from the U1 promoter region, and the U1 PSE is unable to recruit RNA polymerase III to transcribe the U6 gene.Genes coding for most of the small nuclear RNAs (snRNAs) 1 are transcribed by RNA polymerase II, but U6 genes are transcribed by RNA polymerase III. In all cases, however, the promoters of these genes have features that are very similar to each other yet distinct from "classical" RNA polymerase II and RNA polymerase III promoters. In most organisms studied, transcription of U1, U2, U4, and U5 genes by RNA polymerase II or U6 genes by RNA polymerase III requires a unique proximal sequence element (PSE) that is located upstream of position Ϫ40 relative to the transcription start site (Fig. 1A) (1-12).In vertebrates, the PSEs of U1 and U2 genes are functionally interchangeable with the PSEs of U6 genes (13,14). That is, if the PSE of the U6 promoter is replaced with the U1 or U2 PSE, there is no effect on the RNA polymerase III specificity of the U6 promoter. Likewise, the U6 PSE can functionally substitute for the U1 or U2 PSE in the vertebrate U1 and U2 promoters for transcription by RNA polymerase II. In echinoderms and plants, the U1, U2, and U6 PSEs (called USEs in plants) are similarly functionally interchangeable with each other (15-17).The RNA polymerase III specificity of vertebrate U6 snRNA genes is determined by the presence of a TATA box at a fixed distance downstream of the PSE (2, 13, 18). Paradoxically, the promoters of the vertebrate snRNA genes transcribed by RNA polymerase II lack TATA boxes (Fig. 1A). In plants, on the other hand, snRNA genes transcribed by both RNA polymerases have essential TATA boxes in their promoters. In this case, the choice of RNA polymerase is determined by a 10-base pair difference in the spacing between the USE and the respective TATA box (Fig. 1A) (16,17,19).In contrast to the organisms described above, in vitro experiments carried ou...
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