The small nuclear RNA (snRNA)-activating protein complex (SNAPc) is essential for transcription of genes coding for the snRNAs (U1, U2, etc.). In Drosophila melanogaster, the heterotrimeric DmSNAPc recognizes a 21-bp DNA sequence, the proximal sequence element A (PSEA), located approximately 40 to 60 bp upstream of the transcription start site. Upon binding the PSEA, DmSNAPc establishes RNA polymerase II preinitiation complexes on U1 to U5 promoters but RNA polymerase III preinitiation complexes on U6 promoters. Minor differences in nucleotide sequence of the U1 and U6 PSEAs determine RNA polymerase specificity; moreover, DmSNAPc adopts different conformations on these different PSEAs. We have proposed that such conformational differences in DmSNAPc play a key role in determining the different polymerase specificities of the U1 and U6 promoters. To better understand the structure of DmSNAPc-PSEA complexes, we have developed a novel protocol that combines site-specific protein-DNA photo-cross-linking with site-specific chemical cleavage of the protein. This protocol has allowed us to map regions within each of the three DmSNAPc subunits that contact specific nucleotide positions within the U1 and U6 PSEAs. These data help to establish the orientation of each DmSNAPc subunit on the DNA and have revealed cases in which different domains of the subunits differentially contact the U1 versus U6 PSEAs.The Drosophila melanogaster small nuclear RNA (snRNA)-activating protein complex (DmSNAPc) is a heterotrimeric transcription factor (21) that is required for the synthesis of the U1, U2, U4, U5, and U6 spliceosomal snRNAs (2, 22, 31). Homologous protein complexes are required for snRNA gene expression in humans (1,11,12,18,(33)(34)(35) and for spliced leader RNA synthesis in trypanosomes (6,7,30). This indicates that SNAPc appeared early in eukaryotic evolution and continues in contemporary times to be utilized for the transcription of important noncoding RNA molecules in diverse organisms. DmSNAPc binds sequence-specifically to an essential, conserved ϳ21-bp promoter element termed the proximal sequence element A (PSEA) that is located approximately 40 to 60 bp upstream of the transcription start site of fly snRNA genes (8,13,19,23).In animals, the U1 to U5 snRNA genes are transcribed by RNA polymerase II (Pol II), but U6 snRNA genes are transcribed by RNA Pol III (4,5,10,14,19,24,29,31). Surprisingly, the primary determinant of the RNA polymerase specificity of the D. melanogaster snRNA genes is the precise sequence of the PSEA. A few conserved nucleotide differences in the 3Ј half of the 21-bp PSEA are sufficient to determine the polymerase specificity of the fly snRNA genes in vitro and to restrict the polymerase specificity in vivo (2,19,20,26).The three subunits of DmSNAPc are termed DmSNAP190, DmSNAP50, and DmSNAP43 so that the names correspond to the most widely used nomenclature for the three orthologous human SNAPc subunits, SNAP190, SNAP50, and SNAP43. These three human subunits are also known as PTF␣, PTF␤, an...

this paper, we have become aware of a mistake in the identity of the photo-cross-linking probe used to map the domain of DmSNAP190 that cross-linked to phosphate position 24 of the U1 proximal sequence element A (PSEA). The probe used to generate the data presented in Fig. 2 (lanes 1 to 3) and 3C (lanes 13 to 18) was in fact not a position 24 probe; instead, a U1 PSEA phosphate position 12 probe was used in error. Reperformance of these experiments with the correct probe revealed that phosphate position 24 of the U1 PSEA cross-linked to a region of DmSNAP190 located between amino acid residues 169 and 247 rather than between residues 306 and 358. None of the other conclusions of the publication were affected. We sincerely regret this error and any inconvenience that may have resulted to our colleagues.Page 2414, Fig. 2, lanes 1-3: Although this panel was generated with the misidentified probe, the results of the experiment were the same when performed with the correct probe for U1 PSEA position 24 (data not shown). The conclusion that phosphate position 24 cross-linked to the N-terminal half of DmSNAP190 was not affected.Page 2416: Figure 3C, lanes 13-18, should appear as shown below. These results indicate that position 24 of the U1 PSEA cross-linked to DmSNAP190 C terminal of amino acid residue 169 but N terminal of residue 247 inclusive.

Background: SNAP190, the largest subunit of the snRNA-activating protein complex (SNAPc), interacts with DNA via 4.5 Myb repeats. Results: Each Myb repeat was mapped on U1 gene promoter DNA by site-specific protein-DNA photo-cross-linking. Conclusion:The N-terminal repeats contacted DNA nearer the transcription start site, whereas the C-terminal repeats interacted farther upstream. Significance: Structural insights were obtained into SNAPc bound to snRNA gene promoter sequences.

In metazoans, U6 small nuclear RNA (snRNA) gene promoters utilize a proximal sequence element (PSE) recognized by the small nuclear RNA-activating protein complex (SNAPc). SNAPc interacts with the transcription factor TFIIIB, which consists of the subunits TBP, Brf1 (Brf2 in vertebrates), and Bdp1. Here, we show that, in Drosophila melanogaster, DmSNAPc directly recruits Bdp1 to the U6 promoter, and we identify an 87-residue region of Bdp1 involved in this interaction. Importantly, Bdp1 recruitment requires that DmSNAPc be bound to a U6 PSE rather than a U1 PSE. This is consistent with the concept that DmSNAPc adopts different conformations on U6 and U1 PSEs, which lead to the subsequent recruitment of distinct general transcription factors and RNA polymerases for U6 and U1 gene transcription.

The small nuclear RNA (snRNA) activating protein complex (SNAPc) is essential for transcription of genes that encode the snRNAs. Drosophila melanogaster SNAPc (DmSNAPc) consists of three subunits (DmSNAP190, DmSNAP50 and DmSNAP43) that form a stable complex that recognizes an snRNA gene promoter element called the PSEA. Although all three subunits are required for sequence-specific DNA binding activity, only DmSNAP190 possesses a canonical DNA binding domain consisting of 4.5 tandem Myb repeats homologous to the Myb repeats in the DNA binding domain of the Myb oncoprotein. In this study, we use site-specific protein–DNA photo-cross-linking technology followed by site-specific protein cleavage to map domains of DmSNAP190 that interact with specific phosphate positions in the U6 PSEA. The results indicate that at least two DmSNAP190 Myb repeats contact the DNA in a significantly different manner when DmSNAPc binds to a U6 PSEA versus a U1 PSEA, even though the two PSEA sequences differ at only 5 of 21 nucleotide positions. The results are consistent with a model in which the specific DNA sequences of the U1 and U6 PSEAs differentially alter the conformation of DmSNAPc, leading to the subsequent recruitment of different RNA polymerases to the U1 and U6 gene promoters.

Small nuclear RNA activating protein complex (SNAPc) is a multi-subunit transcription factor required for expression of small nuclear RNA genes. This protein binds to a promoter element located approximately 40-65 bp upstream of the transcription start site. In Drosophila melanogaster, DmSNAPc contains three distinct polypeptide subunits: DmSNAP190, DmSNAP50, and DmSNAP43. The subunit stoichiometry in SNAPc complexed with DNA has not been examined. Therefore, the ability of differently tagged but otherwise identical subunits to associate with each other into the same protein-DNA complex was assayed by antibody super-shift analysis. The results reveal that DmSNAPc contains only a single copy of each of the three subunits. Structured summary:MINT-6788068: DmSNAP43 (uniprotkb:Q9VF25), DmSNAP50 (uniprotkb: Q7JUY8) and DmSNAP190 (uniprotkb:Q9VHX0) physically interact (MI:0218) by electrophoretic mobility shift assay (MI:0413) MINT-6788151: DmSNAP190 (uniprotkb:Q9VHX0), DmSNAP50 (uniprotkb: Q7JUY8) and DmSNAP43 (uniprotkb:Q9VF25) physically interact (MI:0218) by molecular sieving (MI:0071) 35 Ó

Edited by Ivan SadowskiRNA polymerase III-transcribed U6 snRNA genes have gene-external promoters that contain TATA boxes. U6 TATA sequences are bound by TFIIIB that in Drosophila contains the three subunits TBP, Brf1, and Bdp1. The overall structure of TFIIIB is still not well understood. We have therefore studied the mode of TFIIIB binding to DNA by site-specific protein-DNA photo-cross-linking. The results indicate that a portion of Brf1 is sandwiched between Bdp1 and TBP upstream of the TATA box. Furthermore, Bdp1 traverses the DNA under the N-terminal stirrup of TBP to interact with the DNA (and very likely Brf1) downstream of the TATA sequence.Keywords: Bdp1; Brf1; Protein-DNA photo-cross-linking; RNA polymerase III transcription; TATA box-binding protein; TFIIIB Eukaryotic RNA polymerase III (Pol III) synthesizes a wide variety of small RNA molecules involved in diverse metabolic processes, including components of the protein synthesis apparatus (tRNA and 5S rRNA) and the spliceosome (U6 snRNA). The Pol III promoters can be divided into three distinct types: Type I (5S rRNA) and Type II (e.g. tRNA) promoters are gene-internal and usually are TATA-less [1][2][3]. Type III promoters (e.g., U6) on the other hand are geneexternal and inevitably contain a TATA sequence [1][2][3][4][5][6][7]. The only general transcription factor that is required at all three types of Pol III promoters is TFIIIB. TFIIIB is considered to be the Pol III initiation factor per se because once bound to the DNA, TFIIIB is able to recruit Pol III for multiple rounds of transcription initiation [8].TFIIIB is composed of three subunits, normally the TATA box-binding protein (TBP), Brf1, and Bdp1 [3]. However, heterogeneity can exist in TFIIIB at different Pol III promoters. For example, in Drosophila melanogaster, the TBP-related factor TRF1 was found to be utilized for Pol III transcription in place of TBP [9]. Recent work in our lab confirmed that TRF1 replaced TBP as a subunit of TFIIIB for tRNA gene transcription in fruit flies. Interestingly, however, we also found that transcription of U6 snRNA genes by Pol III instead utilized TBP rather than TRF1 [10]. Thus, different types of Pol III promoters in insects can differentially utilize either TBP (at Type III promoters) or TRF1 (at Type I and Type II promoters) as a component of TFIIIB.Brf1, another subunit of TFIIIB, is related to the Pol II general transcription factor TFIIB. The N-terminal half of Brf1 is homologous to the zinc ribbon domain and the two cyclin-fold repeats of TFIIB, but Brf1 has an extended C-terminus that averages over 360 amino acid residues in length among yeast, fruit flies, and humans [3].Interestingly, U6 transcription in mammals requires a unique and distinct form of Brf known as Brf2 [11,12]. Human Brf2 contains an N-terminal domain homologous to those found in TFIIB and Brf1, and it Abbreviations TFIIIB, transcription factor IIIB; TBP, TATA box binding protein; TRF1; TBP-related factor 1; TFIIB, transcription factor IIB; Brf1, TFIIB-related factor 1; Brf...

The small nuclear RNA activating protein complex (SNAPc) is a transcription factor essential for expression of genes coding for the spliceosomal snRNAs. In D. melanogaster, DmSNAPc recognizes a 21 base‐pair DNA sequence, the PSEA, located approximately 40–60 base pairs upstream of the transcription start site. DmSNAPc is a heterotrimer of subunits named DmSNAP190, DmSNAP50, and DmSNAP43. All three subunits are required for DNA binding activity, but only DmSNAP190 contains a “canonical” DNA binding domain that consists of 4.5 Myb repeats. Myb repeats are each ~50 amino acid residues in length and were first identified in the DNA binding domain of the Myb oncoprotein. By using a site‐specific protein‐DNA photo‐cross‐linking assay combined with site‐specific protein digestion, we have been able to determine where each of the 4.5 Myb repeats of DmSNAP190 contact the DNA of the U1 PSEA. The protein is arranged with the more C‐terminal Myb repeats contacting the upstream region of the PSEA and the N‐terminal repeats contacting the PSEA closer to the start site. Moreover, the C‐terminal repeats appear to contact the 5′ half of the PSEA in a manner consistent with those observed for Myb‐DNA complexes, whereas the N‐terminal repeats appear to contact the 3′ half of the PSEA (where DmSNAP50 and DmSNAP43 also bind to the PSEA) in a non‐canonical manner. (Supported by NSF and in part by the California Metabolic Research Foundation.)