A Map of Drosophila melanogaster Small Nuclear RNA-activating Protein Complex (DmSNAPc) Domains Involved in Subunit Assembly and DNA Binding

Hung, Ko-Hsuan; Titus, M. Brandon; Chiang, Shu-Chi; Stumph, William E.

doi:10.1074/jbc.m109.027961

Cited by 14 publications

(41 citation statements)

References 28 publications

(84 reference statements)

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“…Electrophoretic mobility shift assays were carried out to confirm the DNAbinding activity of the FLAG-purified DmSNAPc variants (16). Although the results were not quantitative, no significant differences in the DNA-binding activity of the point mutants or of the tagged variants were apparent compared to that of wild-type DmSNAPc (data not shown).…”

Section: Methodsmentioning

confidence: 99%

“…The preparation of untagged and N-and C-terminally FLAG-tagged constructs encoding each of the three DmSNAPc subunits has been recently described (16). Constructs with point mutations to eliminate or introduce hydroxylamine cleavage sites (asparaginyl-glycyl peptide bonds) were prepared by using Stratagene's QuikChange II site-directed mutagenesis kit.…”

Section: Methodsmentioning

confidence: 99%

“…Various forms of the three subunits of DmSNAPc were coexpressed in stably transfected S2 cells by using Invitrogen's Drosophila expression system as previously described (16,21,22). For mapping domains within DmSNAP190, N-or C-terminally tagged DmSNAP190 was coexpressed with untagged DmSNAP50 and DmSNAP43, whereas untagged DmSNAP190 was coexpressed with untagged DmSNAP50 and N-terminally tagged DmSNAP43 (to allow FLAG purification).…”

Section: Methodsmentioning

confidence: 99%

“…Subunit expression was induced with copper sulfate, and DmSNAPc was purified by FLAG immunoaffinity chromatography as recently described (16). Electrophoretic mobility shift assays were carried out to confirm the DNAbinding activity of the FLAG-purified DmSNAPc variants (16).…”

Section: Methodsmentioning

confidence: 99%

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Identification of SNAPc Subunit Domains That Interact with Specific Nucleotide Positions in the U1 and U6 Gene Promoters

Kim

Kang

Lai

et al. 2010

Molecular and Cellular Biology

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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...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Identification of SNAPc Subunit Domains That Interact with Specific Nucleotide Positions in the U1 and U6 Gene Promoters

Kim

Kang

Lai

et al. 2010

Molecular and Cellular Biology

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“…Another Myb-like protein, SNAP190, has been shown to interact with the RNA polymerase II and RNA polymerase III transcription machinery that transcribes the human U1-U5 and U6 small nuclear RNA (snRNA) genes, respectively. These genes contain a proximal sequence element (PSE) that is sufficient to direct basal transcription (59) and that SNAP190 directly contacts (27,29,71). M3BP and SNAP190 have no significant sequence similarities other than at the Myb domains, and BLAST analyses do not indicate these proteins to be homologous.…”

Section: Discussionmentioning

confidence: 99%

Novel Core Promoter Elements and a Cognate Transcription Factor in the Divergent Unicellular Eukaryote Trichomonas vaginalis

Smith

Chudnovsky²,

Simões-Barbosa³

et al. 2011

Molecular and Cellular Biology

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A highly conserved DNA initiator (Inr) element has been the only core promoter element described in the divergent unicellular eukaryote Trichomonas vaginalis, although genome analyses reveal that only ϳ75% of protein-coding genes appear to contain an Inr. In search of another core promoter element(s), a nonredundant database containing 5 untranslated regions of expressed T. vaginalis genes was searched for overrepresented DNA motifs and known eukaryotic core promoter elements. In addition to identifying the Inr, two elements that lack sequence similarity to the known protein-coding gene core promoter, motif 3 (M3) and motif 5 (M5), were identified. Mutational and functional analyses demonstrate that both are novel core promoter elements. M3 [(A/G/T)(A/G)C(G/C)G(T/C)T(T/A/G)] resembles a Myb recognition element (MRE) and is bound

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TFIIIB subunit locations on U6 gene promoter DNA mapped by site‐specific protein–DNA photo‐cross‐linking

Kang

Stumph

2016

FEBS Letters

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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...

show abstract

A Map of Drosophila melanogaster Small Nuclear RNA-activating Protein Complex (DmSNAPc) Domains Involved in Subunit Assembly and DNA Binding

Cited by 14 publications

References 28 publications

Identification of SNAPc Subunit Domains That Interact with Specific Nucleotide Positions in the U1 and U6 Gene Promoters

Identification of SNAPc Subunit Domains That Interact with Specific Nucleotide Positions in the U1 and U6 Gene Promoters

Novel Core Promoter Elements and a Cognate Transcription Factor in the Divergent Unicellular Eukaryote Trichomonas vaginalis

TFIIIB subunit locations on U6 gene promoter DNA mapped by site‐specific protein–DNA photo‐cross‐linking

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