In Vitro Transcription of a <i>Drosophila</i> U1 Small Nuclear RNA Gene Requires TATA Box-Binding Protein and Two Proximal <i>cis</i>-Acting Elements with Stringent Spacing Requirements

Zamrod, Zulkeflie; Tyree, Curtis M.; Song, Yuru; Stumph, William E.

doi:10.1128/mcb.13.9.5918-5927.1993

Cited by 14 publications

(1 citation statement)

References 55 publications

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“…Transcription of genes encoding the spliceosomal small nuclear RNAs (snRNAs) in higher eukaryotes is dependent upon a unique and essential proximal sequence element (PSE) at a conserved location ∼40-65 bp upstream of the transcription start site (1)(2)(3)(4)(5)(6)(7)(8)(9). The PSE is required for transcription of U1, U2, U4 and U5 snRNA genes by RNA polymerase II as well as U6 snRNA genes by RNA polymerase III (1,2,(10)(11)(12).…”

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confidence: 99%

Similarities and differences in the conformation of protein-DNA complexes at the U1 and U6 snRNA gene promoters

Hardin

Ortler

McNamara-Schroeder

et al. 2000

Nucleic Acids Research

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Most small nuclear RNAs (snRNAs) are synthesized by RNA polymerase II, but U6 snRNA is synthesized by RNA polymerase III. In the fruit fly Drosophila melanogaster the RNA polymerase specificity of the snRNA genes is determined by a few nucleotide differences within the proximal sequence element (PSE), a conserved sequence located approximately 40-65 bp upstream of the transcription start site. The PSE is essential for transcription of both RNA polymerase II-transcribed and RNA polymerase III-transcribed snRNA genes and is recognized in Drosophila by a multi-subunit protein factor termed DM:PBP. Previous studies that employed site-specific protein-DNA photocrosslinking indicated that the conformation of the DNA-protein complex is different depending upon whether DM:PBP is bound to a U1 or U6 PSE sequence. These conformational differences of the complex probably represent an early step in determining the selection of the correct RNA polymerase. We have now obtained evidence that DM:PBP modestly bends the DNA upon interacting with the PSE and that the direction of DNA bending is similar for both the U1 and U6 PSEs. Under the assumption that DM:PBP does not significantly twist the DNA, the direction of the bend in both cases is toward the face of the DNA helix contacted by the 45 kDa subunit of DM:PBP. Together with data from partial proteolysis assays, these results indicate that the conformational differences in the complexes of DM:PBP with the U1 and U6 PSEs more likely occur at the protein level rather than at the DNA level.

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Section: Introductionmentioning

confidence: 99%

Similarities and differences in the conformation of protein-DNA complexes at the U1 and U6 snRNA gene promoters

Hardin

Ortler

McNamara-Schroeder

et al. 2000

Nucleic Acids Research

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Transcriptional regulation of snRNAs and its significance for plant development

Ohtani

2016

J Plant Res

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Small nuclear RNA (snRNA) represents a distinct class of non-coding RNA molecules. As these molecules have fundamental roles in RNA metabolism, including pre-mRNA splicing and ribosomal RNA processing, it is essential that their transcription be tightly regulated in eukaryotic cells. The genome of each organism contains hundreds of snRNA genes. Although the structures of these genes are highly diverse among organisms, the trans-acting factors that regulate snRNA transcription are evolutionarily conserved. Recent studies of the Arabidopsis thaliana srd2-1 mutant, which is defective in the snRNA transcription factor, provide insight into the physiological significance of snRNA regulation in plant development. Here, I review the current understanding of the molecular mechanisms underlying snRNA transcription.

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RNA polymerase II/III transcription specificity determined by TATA box orientation.

Wang

Stumph

1995

Proc. Natl. Acad. Sci. U.S.A.

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The TATA box sequence in eukaryotes is located about 25 bp upstream of many genes transcribed by RNA polymerase II (Pol II) and some genes transcribed by RNA polymerase III (Pol III). The TATA box is recognized in a sequence-specific manner by the TATA box-binding protein (TBP), an essential factor involved in the initiation of transcription by all three eukaryotic RNA polymerases. We These and other observations prompted us to investigate whether the orientation of the TATA box, in the absence of any additional promoter elements, could be a primary determinant of RNA polymerase specificity. Indeed, by utilizing a strongThe publicatidn costs of this article were defrayed in part by page charge payaent. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.canonical TATA box sequence and a nuclear extract with similar amounts of Pol II and Pol III activity, we find that a "forward" TATA box specifically directs Pol II transcription, whereas a "reverse" TATA box specifically directs Pol III transcription. MATERIAL AND METHODSConstruction of Templates. The D "Forward" TATA and D "Reverse" TATA templates (shown in Fig. 1) were constructed in two steps. First, synthetic DNA sequences that represent a hybrid of the Drosophila Ul and U6 snRNA gene transcription start sites (11,12) were inserted between the BamHI and Xba I restriction sites of the polylinker of pUC18. After isolation of this recombinant plasmid, synthetic oligonucleotides that contained the TATA sequence in either the forward or reverse orientation were inserted between the Kpn I and BamHI restriction sites of the polylinker.To construct the H/D "Forward" TATA and H/D "Reverse" TATA templates (shown in Fig. 2), synthetic oligonucleotides containing a combination of DNA sequences near the transcription start sites of human Ul, U2, and U6 snRNA genes (8,13,14) were inserted between the EcoRI and Kpn I restriction sites of the D "Forward" TATA and D "Reverse" TATA templates. This placed the human sequences in the opposite orientation and in an upstream position relative to the Drosophila sequences.To construct the pUC18 "Forward" TATA and pUC18 "Reverse" TATA templates (shown in Fig. 3), the Drosophilalike sequences were deleted from the D "Forward" TATA and D "Reverse" TATA templates by digestion with BamHI and Sal I. After filling in the overhanging ends with the Klenow fragment of DNA polymerase, the plasmids were recircularized. Plasmids were grown in Escherichia coli TOP10 cells (Invitrogen), purified by using Qiagen (Chatsworth, CA) Plasmid Midi kits, and used as templates for in vitro transcription.In Vitro Transcription Assays. Transcription reactions (25 ,ul final volume) were carried out for 1 h at 25°C with 10 ,ul of soluble nuclear fraction (SNF) prepared from Drosophila embryos (15-17), 5.5 ,ul HEMG buffer [25 mM Hepes, pH 7.6/12.5 mM MgCl2/0.1 mM EDTA/10% (vol/vol) glycerol/ 1.5 mM dithiothreitol] containing 0.1 M KCl, 7.5 ,ul of ribonucleoside triphosphate (rNTP...

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In Vitro Transcription of a Drosophila U1 Small Nuclear RNA Gene Requires TATA Box-Binding Protein and Two Proximal cis-Acting Elements with Stringent Spacing Requirements

Cited by 14 publications

References 55 publications

Similarities and differences in the conformation of protein-DNA complexes at the U1 and U6 snRNA gene promoters

Similarities and differences in the conformation of protein-DNA complexes at the U1 and U6 snRNA gene promoters

Transcriptional regulation of snRNAs and its significance for plant development

RNA polymerase II/III transcription specificity determined by TATA box orientation.

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