U6 snRNA is transcribed by RNA polymerase III (Pol III) and has an external upstream promoter that consists of a TATA sequence recognized by the TBP subunit of the Pol III basal transcription factor IIIB and a proximal sequence element (PSE) recognized by the small nuclear RNA activating protein complex (SNAPc). Previously, we found that Drosophila melanogaster SNAPc (DmSNAPc) bound to the U6 PSE can recruit the Pol III general transcription factor Bdp1 to form a stable complex with the DNA. Here, we show that DmSNAPc-Bdp1 can recruit TBP to the U6 promoter, and we identify a region of Bdp1 that is sufficient for TBP recruitment. Moreover, we find that this same region of Bdp1 cross-links to nucleotides within the U6 PSE at positions that also cross-link to DmSNAPc. Finally, cross-linking mass spectrometry reveals likely interactions of specific DmSNAPc subunits with Bdp1 and TBP. These data, together with previous findings, have allowed us to build a more comprehensive model of the DmSNAPc-Bdp1-TBP complex on the U6 promoter that includes nearly all of DmSNAPc, a portion of Bdp1, and the conserved region of TBP.

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.

In higher eukaryotes, RNA polymerase III (Pol III) promoters at U6 snRNA genes consist of a TATA box, recognized by TFIIIB, and a proximal sequence element (PSE) recognized by the small nuclear RNA activating protein complex (SNAPc). In the fruit fly Drosophila melanogaster, DmSNAPc consists of three subunits DmSNAP190, DmSNAP50, and DmSNAP43; likewise TFIIIB also consists of three subunits, most commonly TBP, Brf1 and Bdp1. At Drosophila tRNA and 5S RNA gene promoters, TBP‐related factor 1 (TRF1) is utilized in place of TBP, but at U6 promoters the canonical TBP is utilized for Pol III transcription (Verma et al. 2013, JBC 288, 27564–27570).Site‐specific protein‐DNA photo‐cross‐linking studies of DmSNAPc and TFIIIB to U6 promoter DNA indicated that Bdp1 is in close proximity to DmSNAP43 and DmSNAP190 on the U6 promoter (Kang et al. 2016, FEBS Lett 590, 1488–1497). This suggested that the interaction between DmSNAPc and TFIIIB may be mediated, at least in part, by Bdp1. We have investigated this further by electrophoretic mobility shift assays (EMSAs). Surprisingly, we found that DmSNAPc, when bound to the U6 PSE, can recruit Bdp1 to the DNA in the absence of TBP and Brf1. Furthermore, EMSAs indicated that the DmSNAPc‐Bdp1 complex, when bound to U6 promoter DNA, can recruit TBP to form a DmSNAPc‐Bdp1‐TBP‐DNA complex of increased stability.In order to understand the protein‐protein interactions taking place, truncation mutations of Bdp1 were used. It was discovered that an area between amino acid 424 and 510 of Bdp1 is required for its recruitment by DmSNAPc. Furthermore, to investigate whether the TATA box is required for Bdp1 and TBP recruitment, the U6 TATA box was mutated to an unrelated sequence. Although mutation of the TATA box interfered with the recruitment of TBP by the DmSNAPc‐Bdp1 complex, the TATA mutation did not prevent the recruitment of Bdp1 by DmSNAPc. Interestingly, the non‐conserved amino‐terminal tail of TBP contributed to the efficiency of TBP recruitment by the DmSNAPc‐Bdp1 complex.A body of previous work from our lab has shown that DmSNAPc binds to U6 (Pol III transcribed) and U1 promoters (Pol II transcribed) in distinct conformations. Interestingly, when we switched the U6 proximal sequence element A (PSEA) to a U1 PSEA by a 5‐nucleotide change, DmSNAPc was unable to recruit Bdp1. This finding suggests that a surface of DmSNAPc that interacts with Bdp1 may be occluded when DmSNAPc binds to a U1 PSEA. It further provides a mechanism for the polymerase specificity seen when comparing the snRNA U1 and U6 genes.Support or Funding InformationThis work was supported by the National Science Foundation and by the California Metabolic Research Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

RNA 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.

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.

In the fruit fly Drosophila melanogaster, RNA polymerase III transcription was found to be dependent not upon the canonical TATA box-binding protein (TBP) but instead upon the TBP-related factor 1 (TRF1) (Takada, S., Lis, J. T., Zhou, S., and Tjian, R. (2000) Cell 101, 459-469). Here we confirm that transcription of fly tRNA genes requires TRF1. However, we unexpectedly find that U6 snRNA gene promoters are occupied primarily by TBP in cells and that knockdown of TBP, but not TRF1, inhibits U6 transcription in cells. Moreover, U6 transcription in vitro effectively utilizes TBP, whereas TBP cannot substitute for TRF1 to promote tRNA transcription in vitro. Thus, in fruit flies, different classes of RNA polymerase III promoters differentially utilize TBP and TRF1 for the initiation of transcription.

U1 and U6 snRNA genes are transcribed by RNA polymerases II and III respectively, yet the transcription of each is dependent upon the small nuclear RNA activating protein complex (SNAPc). 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 of each gene. 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 tandem Myb repeats. Myb repeats are each ~50 amino acid residues in length and were first identified as forming 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 map where each of the 4.5 Myb repeats of DmSNAP190 contact the DNA of the U1 and U6 PSEAs. Although many of the contact points are similar, the Myb repeats of DmSNAP190 interact with the U1 DNA primarily through the major groove, but new and distinct Myb repeats‐DNA contacts that span the minor groove are observed when DmSNAPc binds to a U6 PSEA. This conformational shift in DmSNAP190 binding is likely important in determining the RNA polymerase specificity of the U1 and U6 gene promoters. (Supported by NSF and in part by the California Metabolic Research Foundation.)

Transcription of snRNA genes depends upon the recognition of the proximal sequence element (PSE) by the snRNA activating protein complex SNAPc. In Drosophila melanogaster, all subunits of DmSNAPc (DmSNAP43, DmSNAP50, and DmSNAP190) are required for PSE-binding activity. Previous work demonstrated that a non-canonical DmSNAP43 domain bounded by residues 193-272 was essential for DmSNAPc to bind to the PSE. In this study, the contribution of amino acid residues within this domain to DNA binding by DmSNAPc was investigated by alanine-scanning mutagenesis. The results have identified two clusters of residues within this domain required for the sequence-specific DNA-binding activity of DmSNAPc.

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.)

Myb repeats ∼52 amino acid residues in length were first characterized in the oncogenic Myb transcription factor, which contains three tandem Myb repeats in its DNA-binding domain. Proteins of this family normally contain either one, two, or three tandem Myb repeats that are involved in protein-DNA interactions. The small nuclear RNA (snRNA)-activating protein complex (SNAPc) is a heterotrimeric transcription factor that is required for expression of small nuclear RNA genes. This complex binds to an essential promoter element, the proximal sequence element, centered ∼50 base pairs upstream of the transcription start site of snRNA genes. SNAP190, the largest subunit of SNAPc, uncharacteristically contains 4.5 tandem Myb repeats. Little is known about the arrangement of the Myb repeats in the SNAPc-DNA complex, and it has not been clear whether all 4.5 Myb repeats contact the DNA. By using a site-specific protein-DNA photo-cross-linking assay, we have now mapped specific nucleotides where each of the Myb repeats of Drosophila melanogaster SNAP190 interacts with a U1 snRNA gene proximal sequence element. The results reveal the topological arrangement of the 4.5 SNAP190 Myb repeats relative to the DNA and to each other when SNAP190 is bound to a U1 promoter as a subunit of SNAPc.

The small nuclear RNAs (snRNAs) are an essential class of non-coding RNAs first identified over 30 years ago. Many of the well-characterized snRNAs are involved in RNA processing events. However, it is now evident that other small RNAs, synthesized using similar mechanisms, play important roles at many stages of gene expression. The accurate and efficient control of the expression of snRNA (and related) genes is thus critical for cell survival. All snRNA genes share a very similar promoter structure, and their transcription is dependent upon the same multi-subunit transcription factor, termed the snRNA activating protein complex (SNAPc). Despite those similarities, some snRNA genes are transcribed by RNA polymerase II (Pol II), but others are transcribed by RNA polymerase III (Pol III). Thus snRNA genes provide a unique opportunity to understand how RNA polymerase specificity is determined and how distinct transcription machineries can interact with a common factor. This review will describe efforts taken toward solving those questions by using the fruit fly as a model organism. Drosophila melanogaster SNAPc (DmSNAPc) binds to a proximal sequence element (PSEA) present in both Pol II and Pol III snRNA promoters. Just a few differences in nucleotide sequence in the Pol II and Pol III PSEAs play a major role in determining RNA polymerase specificity. Furthermore, these same nucleotide differences result in alternative conformations of DmSNAPc on Pol II and Pol III snRNA gene promoters. It seems likely that these DNA-induced alternative DmSNAPc conformations are responsible for the differential recruitment of the distinct transcriptional machineries.

The small nuclear RNA activating protein complex (SNAPc) is essential for transcription of genes coding for the spliceosomal snRNAs. In D. melanogaster, the heterotrimeric DmSNAPc recognizes a 21 base-pair DNA sequence, the PSEA, located approximately 40–60 base pairs upstream of the transcription start site. Upon binding the PSEA, DmSNAPc establishes RNA polymerase II pre-initiation complexes on U1-U5 promoters but RNA polymerase III pre-initiation complexes on U6 promoters. Minor differences in nucleotide sequence of the U1 and U6 PSEAs determine the polymerase specificity; moreover, DmSNAPc adopts different conformations on these two different PSEAs. Such conformational differences in DmSNAPc likely play a key role in establishing the RNA polymerase specificities of the U1 and U6 promoters. We have now determined in some cases that distinct sub-domains of the DmSNAP50 and DmSNAP43 subunits are differentially in close contact with different nucleotide positions of the U1 and U6 PSEAs. This was established by site-specific protein-DNA photocrosslinking combined with site-specific chemical digestion of the protein. The third subunit, DmSNAP190, has a unique DNA-binding domain that consists of 4.5 Myb repeats; we are now mapping the architectural arrangement of these Myb repeats on the U1 and U6 PSEAs. (Supported by NSF and in part by the California Metabolic Research Foundation.)

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.

Transcription of genes coding for the small nuclear RNAs (snRNAs) is dependent upon a unique transcription factor known as the small nuclear RNA-activating protein complex (SNAPc). SNAPc binds to an essential proximal sequence element located about 40-65 base pairs upstream of the snRNA transcription start site. In the fruit fly Drosophila melanogaster, DmSNAPc contains three distinct polypeptides (DmSNAP190, DmSNAP50, and DmSNAP43) that are stably associated with each other and bind to the DNA as a complex. We have used mutational analysis to identify domains within each subunit that are involved in complex formation with the other two subunits in vivo. We have also identified domains in each subunit required for sequence-specific DNA binding. With one exception, domains required for subunit-subunit interactions lie in the most evolutionarily conserved regions of the proteins. However, DNA binding by DmSNAPc is dependent not only upon the conserved regions but is also highly dependent upon domains outside the conserved regions. Comparison with functional domains identified in human SNAPc indicates many parallels but also reveals significant differences in this ancient yet rapidly evolving system.

The small nuclear RNA activating protein complex (SNAPc) is an evolutionarily conserved multi‐subunit factor required for transcription of the spliceosomal small nuclear RNA (snRNA) genes by both RNA polymerase II (U1, U2, U4, and U5) and RNA polymerase III (U6). Three distinct polypeptides have been identified as subunits of D. melanogaster SNAPc; however, the stoichiometry of these three subunits in DmSNAPc had not been investigated. By co‐expressing each subunit with two different tags and by doing band‐shift and super‐shift analyses, we have determined that DmSNAPc is a heterotrimer with a 1:1:1 subunit stoichiometry. DmSNAPc recognizes an ~21 bp long DNA sequence denoted the PSEA about 40‐60 bp upstream of the transcription start site. Interestingly, the PSEAs of the U1 and U6 genes are not interchangeable even though they are identical at 16 of 21 nucleotide positions. In fact, changing the U1 PSEA to a U6 PSEA inactivated the U1 promoter. We have now found that this substitution does not affect the association of DmSNAPc with the promoter; instead, it disrupts the recruitment of TBP. This finding is consistent with a working model in which DmSNAPc binds in different conformations to the U1 and U6 PSEAs. We believe these conformational differences in DmSNAPc lead to differential RNA polymerase selectivity at the U1 and U6 promoters. Supported by NSF and in part by the California Metabolic Research Foundation.

The small nuclear RNA activating protein complex (SNAPc) is the major unique transcription factor required for transcription of genes coding for small nuclear RNAs (snRNAs). In the fruit fly Drosophila melanogaster, DmSNAPc contains three distinct subunits (DmSNAP190, DmSNAP50, and DmSNAP43) that form a complex before binding to an snRNA gene promoter. We have used mutational analysis to identify domains within each subunit of DmSNAPc that are required for complex formation with the other two subunits in vivo. Also, we mapped domains in each subunit that are required for the DNA‐binding activity of DmSNAPc. We have found that the most evolutionarily conserved regions of the proteins are involved in SNAP complex assembly. Nevertheless, we found that domains outside of the conserved regions are also important for the DNA binding activity of DmSNAPc, even though they are not required for subunit assembly. Comparing our findings with published results in the human system indicates not only many important similarities but also significant differences in this ancient though rapidly evolving system. This work is supported by National Science Foundation grants MCB‐0131151 and MCB‐0641350 and in part by the California Metabolic Research Foundation. M. T. is a recipient of an Arne N. Wick Pre‐doctoral Research Fellowship from the California Metabolic Research Foundation.

Most of the spliceosomal small nuclear RNAs (U1, U2, U4, and U5) are synthesized by RNA polymerase II, but U6 snRNA is synthesized by RNA polymerase III. In Drosophila melanogaster. transcription of all snRNA genes requires a unique proximal promoter element, the PSEA. Surprisingly, the RNA polymerase specificity of the snRNA genes is determined by a few nucleotide differences between the PSEAs in the two classes of genes. In both classes, the PSEA is recognized by the small nuclear RNA activating protein complex (DmSNAPc). Previous transfection assays demonstrated that Pol II was unable to initiate transcription from a U1 promoter that contained the U6 PSEA. We have now used chromatin immunoprecipitation assays to determine at which step Pol II pre‐initiation complex assembly is disrupted when the U1 promoter contains a U6 PSEA. Interestingly, we found that DmSNAPc stably assembled on a U1 promoter that contained either the U1 or the U6 PSEA. However, although TBP was recruited to the wild type U1 promoter, TBP failed to assemble in vivo on the U1 promoter that contained the U6 PSEA. These results indicate that changing the U1 PSEA to a U6 PSEA had no effect on the binding of DmSNAPc, but it instead suggests that DmSNAPc is in the wrong conformation when bound to a U6 PSEA to recruit TBP to the U1 promoter.N.B. is a recipient of the Arne N. Wick Predoctoral Research Fellowship from the California Metabolic Research Foundation.

Antibody-based assays have been a cornerstone of infectious disease diagnostics for over 100 years [1]. These assays rely on the exquisite sensitivity and specificity of humoral response to almost all infections. While next-generation sequencing (NGS) has tremendous potential to improve diagnostics and uncover host-microbial relationships by directly identifying nucleic acids from infectious microbes, challenges and opportunities for new approaches remain. Here, we review a group of cutting-edge techniques that couple antibody responses with flow cytometry of antibody tagged microbes and NGS. These studies are bringing into focus the dynamic relationship between our immune systems and endogenous microbial communities, which are an important source of pathogens. For simplicity, we use the umbrella term mFLOW-Seq (microbial flow cytometry coupled to NGS) to describe these approaches.