Identification and Topological Arrangement of <i>Drosophila</i> Proximal Sequence Element (PSE)-Binding Protein Subunits That Contact the PSEs of U1 and U6 Small Nuclear RNA Genes

Wang, Yan; Stumph, William E.

doi:10.1128/mcb.18.3.1570

Cited by 28 publications

(92 citation statements)

References 35 publications

(59 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Photo-cross-linking reactions and analysis were carried out as previously described (33). Briefly, derivatized DNA fragments were incubated with partially purified DmPBP, either the HA300 fraction from fruit fly embryo nuclear extracts (29) or a nickel-chelating column fraction of overexpressed His 6 -tagged protein from S2 cells.…”

Section: Methodsmentioning

confidence: 99%

“…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 functional polymerase specificity studies, led us to propose a hypothetical model in which the U1 and U6 PSEAs act as differential allosteric effectors of the conformation of DmPBP, subsequently leading to the specific downstream recruitment of either RNA polymerase II or RNA polymerase III (5,33).…”

mentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

See 3 more Smart Citations

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

Li¹,

Harding

Parise³

et al. 2004

Molecular and Cellular Biology

Self Cite

104

View full text Add to dashboard Cite

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...

show abstract

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

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

Li¹,

Harding

Parise³

et al. 2004

Molecular and Cellular Biology

Self Cite

104

View full text Add to dashboard Cite

show abstract

“…ultimately be determined by different conformations of the same factor, which are dictated by the exact PSEA sequence (12).…”

Section: The Pse Binding Factorsmentioning

confidence: 99%

Small Nuclear RNA Genes: a Model System to Study Fundamental Mechanisms of Transcription

Hernandez

2001

Journal of Biological Chemistry

193

196

View full text Add to dashboard Cite

The human small nuclear RNA (snRNA) 1 genes, which encode snRNAs that are involved in RNA processing reactions such as mRNA splicing, serve as prototypes for a family of genes whose promoters are characterized by the presence of a proximal sequence element (PSE) and a distal sequence element (DSE). From a transcription point of view, this family of genes is highly interesting because all of its members have very similar promoters, even though some of them are transcribed by RNA polymerase (pol) II and others by pol III. As a result, the snRNA genes have served as a model system to explore how RNA polymerase specificity is determined and, in general, to compare the pol II and III transcription machineries. This has led to the concept that the pol II and III transcription machineries use common factors, the best known of which is the TATA box binding protein (TBP). In addition, the relative simplicity of these promoters has also made them an attractive system to study how transcriptional activators perform their function. Fig. 1 shows the structures of snRNA promoters from Homo sapiens (Hs), Arabidopsis thaliana (At), and Drosophila melanogaster (Dm) and serves to illustrate the remarkable fact that although snRNA promoters have diverged during evolution, the close similarity between those recognized by pol II and those recognized by pol III has been conserved. In fact, in each of the examples in Fig. 1, RNA polymerase specificity can be changed by altering a single parameter, indicated in red on the figure. The Structure of snRNA PromotersIn the human genes, the U1 and U2 snRNA promoters serve as the prototypic pol II snRNA promoters, and the U6 snRNA promoter serves as the prototypic pol III snRNA promoter (see Ref. 1 for a review). The human pol II snRNA core promoters contain only one essential element, the PSE, whereas the pol III snRNA core promoters consist of two elements, the PSE and a TATA box located at a fixed distance downstream. The DSE serves to enhance transcription from the core promoter. Both the DSE and the PSE can be interchanged between pol II and III snRNA promoters with no effect on RNA polymerase specificity, which is determined by the presence or absence of the TATA box. The A. thaliana pol II and III snRNA promoters contain an upstream sequence element (USE) and a TATA box, which are both interchangeable between the pol II and III snRNA promoters. RNA polymerase specificity is determined in this case by the exact spacing between the USE and the TATA box, which is 33-34 base pairs (bp) and 23-24 bp in the pol II and III snRNA promoters, respectively (2).The D. melanogaster pol II snRNA promoters contain two elements referred to as the PSEA and the PSEB spaced by 8 bp, and the pol III snRNA promoters contain a PSEA and a TATA box spaced by 12 bp. The PSEA is quite conserved in various pol II and III snRNA promoters, but positions 19 and 20 of the 21-bp elements are always g/aG in the pol II and TC in the pol III snRNA promoters. RNA polymerase specificity is determined by the precise sequ...

show abstract

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

Kang

Stumph

2016

FEBS Letters

Self Cite

View full text Add to dashboard Cite

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

Identification and Topological Arrangement of Drosophila Proximal Sequence Element (PSE)-Binding Protein Subunits That Contact the PSEs of U1 and U6 Small Nuclear RNA Genes

Cited by 28 publications

References 35 publications

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

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

Small Nuclear RNA Genes: a Model System to Study Fundamental Mechanisms of Transcription

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

Contact Info

Product

Resources

About