Architectural Arrangement of Cloned Proximal Sequence Element-Binding Protein Subunits on Drosophila U1 and U6 snRNA Gene Promoters

Abstract

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...

Characterization of a Multisubunit Transcription Factor Complex Essential for Spliced-Leader RNA Gene Transcription in Trypanosoma brucei

“…The protein complex binds to the proximal sequence element of snRNA gene promoters and consists of five subunits, three of which are essential for transcriptional activation, namely SNAP190, SNAP50, and SNAP43. The recent characterization of Drosophila melanogaster SNAP c revealed orthologues to these three proteins but no additional subunits (21). The sequence conservation between human and insect SNAPs is limited, and only functionally important domains exhibit substantial similarity (21).…”

“…The recent characterization of Drosophila melanogaster SNAP c revealed orthologues to these three proteins but no additional subunits (21). The sequence conservation between human and insect SNAPs is limited, and only functionally important domains exhibit substantial similarity (21). In addition to SNAP c , human snRNA gene transcription essentially depends on the basal transcription factors TBP, transcription factor IIA (TFIIA), TFIIB, TFIIF, and TFIIE (15).…”

Differential Utilization of TATA Box-binding Protein (TBP) and TBP-related Factor 1 (TRF1) at Different Classes of RNA Polymerase III Promoters

“…Combinations of the expression constructs described above were used to co-transfect D. melanogaster S2 cells, and stably transfected cell lines were established by selection on blasticidin-containing medium as described previously (26). Lines were established that expressed untagged Brf1 and untagged Bdp1 together along with either tagged TBP or tagged TRF1.…”

Mitotic Functions for SNAP45, a Subunit of the Small Nuclear RNA-activating Protein Complex SNAPc

“…There is a recognizable SNAP c in Drosophila melanogaster, but the complex is limited to the components present in the minimal functional core of SNAP c , mini-SNAP c (27), and lacks SNAP45 as well as the SNAP190 C-terminal third that, in human SNAP c , associates with SNAP45. Thus, SNAP45 seems to have appeared in vertebrates, both as an elaboration of the SNAP complex, and as a protein required for proper mitosis.…”

“…1D, panels [17][18][19][20], which was then concentrated in the mid-body at telophase (Fig. 1D, panels [21][22][23][24][25][26][27][28]. To visualize these structures better, we used methanol fixation.…”