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Previous Article | Next Article 
Molecular and Cellular Biology, August 2005, p. 7303-7313, Vol. 25, No. 16
0270-7306/05/$08.00+0 doi:10.1128/MCB.25.16.7303-7313.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Department of Genetics and Developmental Biology and Department of Molecular, Microbial, and Structural Biology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, Connecticut 06030-3710
Received 15 March 2005/ Returned for modification 28 April 2005/ Accepted 20 May 2005
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Trypanosomatids diverged from the main eukaryotic lineage very early in evolution (11, 34). As a consequence, protein identification in trypanosomatid genome databases is only successful for the most conserved proteins, such as the TATA-binding protein (TBP)-related factor 4 (TRF4). Chromatin immunoprecipitation revealed that TRF4 is associated with SL RNA gene sequences, and an RNA interference analysis indicated that TRF4 functions in SL RNA gene transcription (30). A second factor associated with SL RNA gene transcription was biochemically purified in L. seymouri (2). The factor was termed PBP-1; it consists of three subunits with apparent molecular masses of 57, 46, and 36 kDa, and it binds specifically to the SL RNA gene USE (25). While p36 was not characterized, p46 appeared to be a parasite-specific protein (2). Conversely, p57 was identified as a divergent orthologue of SNAP50, a subunit of the human small nuclear RNA (snRNA)-activating protein complex (SNAPc [reference 9]) also described as a proximal sequence element-binding transcription factor (37), suggesting that PBP-1 is a SNAPc-like factor (2).
Human SNAPc is an essential factor for RNA polymerase II- or III-mediated transcription of snRNA genes (reviewed in reference 10). The protein complex binds to the proximal sequence element of snRNA gene promoters and consists of five subunits, three of which are essential for transcriptional activation, namely SNAP190, SNAP50, and SNAP43. The recent characterization of Drosophila melanogaster SNAPc revealed orthologues to these three proteins but no additional subunits (21). The sequence conservation between human and insect SNAPs is limited, and only functionally important domains exhibit substantial similarity (21). In addition to SNAPc, human snRNA gene transcription essentially depends on the basal transcription factors TBP, transcription factor IIA (TFIIA), TFIIB, TFIIF, and TFIIE (15). Orthologues of these factors may be involved in trypanosome SL RNA gene transcription as well because the 139-nucleotide-long SL RNA belongs to the class of snRNAs. However, except for the TBP homologue TRF4, they have not been identified in trypanosomatid genome databases which are virtually complete for T. brucei and Leishmania major.
We have recently cloned and epitope-tagged TbSNAP50, the T. brucei orthologue of human SNAP50 and L. seymouri p57, and confirmed that this protein binds to the SL RNA gene USE of T. brucei (32). Here, we have created a novel tag combination for tandem affinity purification (TAP) termed the PTP tag. By employing the PTP tagging and purification method initially with TRF4 and subsequently with other subunits, we isolated a stable multisubunit complex which directs SL RNA gene transcription in a cell extract. The complex consists of TRF4, three SNAPc subunits, the T. brucei orthologue of the small TFIIA subunit, and a sixth protein which appears to be an extremely divergent orthologue of the large TFIIA subunit.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-tubulin genes, respectively. In a further development of the original resistance marker cassette (20), we separated the HSP70 intergenic region from NEO-R by an NdeI restriction site and NEO-R from the tubulin flank by BamHI, HpaI, and BstBI restriction sites. This allows precise replacement of the selectable marker gene within the context of pC-PTP-NEO. For PTP tagging of TbTRF4, TbSNAP50, TbSNAP2, and TbTFIIA-2 derivatives of pC-PTP-NEO were generated. In each case, the C-terminal protein-coding region preceding the PTP sequence was exchanged. In pTbTRF4-PTP-NEO, pTbSNAP50-PTP-NEO, pTbSNAP2-PTP-NEO, and pTFIIA-2-PTP-NEO the corresponding gene sequence comprised 783, 462, 692, and 400 bp, respectively. For genomic integration, both SNAP plasmids were linearized with restriction enzyme BstBI, whereas pTbTRF4-PTP-NEO and pTFIIA-2-PTP-NEO were linearized with SnaBI and StuI, respectively. In the construct pPURO-HA-TbTFIIA-1, which was used for N-terminal hemagglutinin (HA) tagging of TFIIA-1, an H23-PURO-T selectable marker cassette precedes a second cassette comprising 677 bp of TbRPA2 5' flank (31), the translation initiation codon, the HA tag, and 478 bp of the TFIIA-1 N-terminal coding region. The template constructs for in vitro transcription assays, SLins 19 and GPEET-trm, have been described previously (7, 19).
Cell culture and generation of cell lines. Cultivation of procyclic forms of T. brucei brucei strain 427 was carried out as described previously (19). For the generation of cell lines expressing epitope-tagged proteins, 10 µg of linearized plasmid was transfected into procyclic 427 cells by electroporation as detailed elsewhere (5). In the cell line TbA3, the second TbSNAP2 allele was knocked out by a PCR product in which the coding region of the hygromycin phosphotransferase was fused to 101 bp of TbSNAP2 5' flank and 102 bp of TbSNAP2 3' flank. Transfected cells were cloned by limiting dilution and selected with 40 µg/ml G418, 20 µg/ml hygromycin, and/or 4 µg/ml puromycin (Sigma, St. Louis, MO). Correct integration of constructs was verified by PCR and Southern analysis in each case, and expression of the epitope-tagged proteins was analyzed by immunoblotting with monoclonal anti-ProtC and anti-HA antibodies (Roche, Indianapolis, IN).
PTP purification. For purification of PTP-tagged proteins, a 2.5-liter culture of procyclic T. brucei cells was grown to a density of 2 x 107 cells per ml, harvested, and extracted as described previously (19). All further steps were carried out at 4°C or on ice. The resulting 6 ml of unconcentrated extract was mixed with 0.5 ml of a 1-ml PA-150 buffer aliquot (150 mM potassium chloride, 20 mM Tris-HCl, pH 7.7, 3 mM MgCl2, 0.5 mM dithiothreitol, 0.1% Tween 20) in which a Complete Mini, EDTA-free protease inhibitor cocktail tablet (Roche, Indianapolis, IN) was dissolved. Subsequently, the extract was added to a 200-µl settled bead volume of immunoglobulin G (IgG) Sepharose 6 Fast Flow beads (Amersham Biosciences, Piscataway, NJ) and equilibrated with PA-150 buffer in a 0.8- by 4-cm Poly-Prep chromatography column (Bio-Rad, Hercules, CA). PTP-tagged proteins were bound to IgG Sepharose by rotating the closed column for 2 h. Subsequently, the flowthrough was collected by gravity flow and beads were washed with 25 ml of PA-150 buffer. After equilibrating the beads in 15 ml of tobacco etch virus (TEV) protease buffer (PA-150 with 0.5 mM EDTA), they were resuspended in 2 ml of TEV protease buffer containing 300 units of AcTEV protease (Invitrogen, Carlsbad, CA) and rotated overnight. The TEV protease eluate was collected by gravity flow, diluted to 6 ml by a wash of the IgG Sepharose beads with 4 ml of PC-150 buffer (PA-150 buffer containing 1 mM calcium chloride), and mixed with the remaining 0.5 ml of the protease inhibitor cocktail. For anti-ProtC affinity purification, calcium chloride was added to the TEV protease eluate to a final concentration of 2 mM, which was then combined in a new column with a 200-µl settled bead volume of anti-protein C affinity matrix (Roche, Indianapolis, IN) equilibrated in PC-150 buffer. The column was rotated for 2 h, after which the flowthrough was collected and the matrix washed with 60 ml of PC-150. Finally, ProtC-tagged proteins were eluted either with EGTA elution buffer (5 mM Tris-HCl, pH 7.7, 10 mM EGTA, 5 mM EDTA, 10 µg/ml leupeptin) or with peptide elution buffer (transcription buffer containing 0.5 mg/ml ProtC peptide and 0.1% Tween 20). In case of EGTA elution, five consecutive steps were carried out in which the beads were resuspended in 0.6 ml of EGTA elution buffer and rotated for 15 min at room temperature. For peptide elution, the beads were mixed with 300 µl of peptide elution buffer and rotated at room temperature for 1 h.
The peptide eluate was used without further concentration in functional assays. The volume of the EGTA eluate was reduced from 3 ml to approximately 600 µl by evaporation in a vacuum concentrator. Subsequently, the proteins were bound to 10 µl of the hydrophobic StrataClean resin (Stratagene, La Jolla, CA), released into sodium dodecyl sulfate (SDS) loading buffer at 80°C, separated on SDS-polyacrylamide gels, and Coomassie stained with the GelCode blue stain reagent (Pierce, Rockford, IL). Proteins were identified by liquid chromatography-tandem mass spectrometry (see the list of identified peptides for each protein in the supplemental material).
Western detection. In immunoblot analyses, proteins were separated on SDS-polyacrylamide gels, electroblotted onto polyvinylidene difluoride membrane, and detected either by the protein A-specific PAP reagent (Sigma, St. Louis, MO), the anti-ProtC antibody HPC4, or a monoclonal anti-HA antibody in combination with the BM chemiluminescence blotting substrate (Roche, Indianapolis, IN). For detection of TbSNAP3, a polyclonal antibody against the peptide EMRRRINTESLLKRK was raised.
Sedimentation on sucrose gradients. Sedimentation analysis was carried out by ultracentrifugation of 3.8-ml 10 to 40% linear sucrose gradients containing 20 mM HEPES-KOH, pH 7.7, 20 mM potassium L-glutamate, 3 mM MgCl2, and 20 or 400 mM potassium chloride. Gradients were overlaid with 200 µl of TbSNAP2-P eluate and centrifuged at 42,000 rpm in a Beckman SW55 rotor for 19 h at 4°C. Twenty fractions were collected from top to bottom, and protein was precipitated using StrataClean resin as described above. Proteins were separated on 12% SDS-polyacrylamide gels and visualized by silver staining using the Silver Stain Plus kit (Bio-Rad, Hercules, CA) according to the manufacturer's protocol.
Promoter pull-down and in vitro transcription assays. In vitro transcription reactions and promoter pull-down assays were carried out as described in detail elsewhere (18, 32). In brief, transcription reactions were carried out in a volume of 40 µl for 60 min at 27°C and the reaction mixtures contained 8 µl of extract, 20 mM potassium L-glutamate, 20 mM KCl, 3 mM MgCl2, 20 mM HEPES-KOH, pH 7.7, 0.5 mM of each nucleoside triphosphate (NTP), 20 mM creatine phosphate, 0.48 mg ml–1 of creatine kinase, 2.5% polyethylene glycol, 0.2 mM EDTA, 0.5 mM EGTA, 4 mM dithiothreitol, 10 mg ml–1 leupeptin, 10 mg ml–1 aprotinin, and 40 µg ml–1 of exogenously added DNA. The latter comprised 7.5 µg ml–1 of SLins19 template, 20 µg ml–1 of GPEET-trm template, and 12.5 µg ml–1 of vector DNA. In addition, each reaction mixture contained 0.1 mg ml–1 of ProtC peptide to exclude the possibility of a nonspecific peptide effect. To specifically detect GPEET-trm and SLins19 RNAs, total RNA was prepared from each reaction and analyzed by extension of 32P-end-labeled primers Tag_PE (19) and SLtag (7), which are complementary to unrelated oligonucleotide tags of GPEET-trm and SLins19, respectively. The primer extension products were separated on 6% polyacrylamide-50% urea gels and visualized by autoradiography.
For the pull-down assays, biotinylated promoter DNA fragments were generated by PCR using a 5'-biotinylated sense oligonucleotide. For each reaction, 500 ng of biotinylated DNA fragments was coupled to 10 µl (100 µg) of RNase-free, paramagnetic M-280 streptavidin Dynabeads (Dynal) according to the manufacturer's protocol. The beads were equilibrated and blocked for 30 min at room temperature in TK20 buffer (150 mM sucrose, 20 mM HEPES-KOH, pH 7.7, 20 mM potassium L-glutamate, 20 mM KCl, 3 mM MgCl2, 2.5% [wt/vol] polyethylene glycol, 0.2 mM EDTA, 0.5 mM EGTA, 4 mM dithiothreitol, 10 µg ml–1 leupeptin, 10 µg ml–1 aprotinin) containing 5 mg ml–1 bovine serum albumin and 5 mg ml–1 polyvinylpyrrolidone. Subsequently, the beads were washed twice with 0.5 ml of TK20 buffer and incubated in a 40-µl in vitro transcription reaction mixture for 15 min on ice and for 15 min at 27°C. Beads were washed three times with 0.5 ml TK20 buffer and once with 0.5 ml of TN40 buffer (150 mM sucrose, 20 mM Tris pH 8.0, 40 mM NaCl, 3 mM MgCl2, 0.5 mM dithiothreitol, 10 µg ml–1 leupeptin, 10 µg ml–1 aprotinin) before proteins were eluted in standard SDS gel loading buffer by a 5-min-long incubation at 70°C.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 nM) and, as a unique property, has a calcium binding site which needs to be occupied for epitope binding (35). To facilitate efficient PTP tagging of trypanosome proteins, we designed the genome integration vector pC-PTP-NEO, which allows fusion of a C-terminal protein-coding region to the PTP tag sequence in a single cloning step (Fig. 1B). Cell lines which stably express PTP-tagged proteins were established by targeted insertion of the vector to the gene of interest (Fig. 1C).
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- and ß-tubulin and of IgG heavy chain (Fig. 2C) (data not shown).
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The major 55-kDa protein was identified as TbSNAP50, indicating that TRF4 interacts with TbSNAPc. The other four major proteins were new discoveries and are described here for the first time. The protein with an apparent size of 26 kDa was unambiguously identified by sequence homology to be an orthologue of human and insect SNAP43 (Fig. 3A). The protein consists of 234 amino acids and has a predicted size of 26.1 kDa. A comparison between human and insect SNAP43 had shown that only the N-terminal region is conserved (21) due to its interacting function with SNAP50 (24). Correspondingly, sequence conservation in the 26-kDa protein is restricted to the N terminus. We named this protein TbSNAP3 because of the finding of another SNAP of larger size (see below) and because of its deviant size in comparison to its human orthologue. Interestingly, TbSNAP3 is 10 kDa smaller than the smallest yet uncharacterized SNAPc subunit of L. seymouri (25). We have identified the gene sequences encoding the TbSNAP3 orthologues in the genome databases of T. cruzi (LM.36.1.Contig1; Sanger Center, Cambridge, United Kingdom) and L. major (t_cruzi chr_0 1047053510181 5857; TIGR, Rockville, MD). While the predicted sizes of the T. cruzi protein TcSNAP3 and TbSNAP3 are similar, the L. major protein LmSNAP3 has a predicted size of 38 kDa, in accordance with the apparent size of the L. seymouri protein. As revealed by a sequence alignment, the larger size of LmSNAP3 is due to an internal domain which is lacking in the trypanosome proteins (Fig. 3A) (data not shown).
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Finally, to prove correct identification of TbSNAP2 and TbSNAP3, we HA tagged either protein in cells expressing TbSNAP50-PTP and showed that both proteins copurified with TbSNAP50, in contrast to an unrelated HA-tagged protein (Fig. 4, compare lanes 3, 6, and 9).
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subunit in higher eukaryotes and as TOA2 in the budding yeast Saccharomyces cerevisiae. The conservation of this protein is sufficient for an unambiguous assignment, and therefore it was termed TbTFIIA-2 (Fig. 5A). As has been analyzed in yeasts and mammals, TFIIA is a basal factor for RNA polymerase II-mediated transcription (26) and absolutely essential for snRNA gene transcription (15). While yeast TFIIA consists of the two subunits TOA1 and TOA2, the larger subunit of higher eukaryotes is proteolytically cleaved into and ß subunits. The bi- and tripartite nature of TFIIA in other eukaryotes strongly argues that the remaining major protein which copurified with TRF4-P is the orthologue of TOA1/TFIIA-ß. However, the corresponding T. brucei protein which was identified from the 75-kDa protein band is the least conserved among the identified proteins. While a putative orthologue with 22.3%/34.2% sequence identity/similarity could be identified in the T. cruzi genome database (t_cruzi chr_0 1047053508897 7919; The Institute for Genomic Research [TIGR], Rockville, MD), no convincingly conserved sequence was obtained from the database of the more distantly related L. major. Among TOA1 and TFIIA-ß sequences, only the 50 N-terminal residues and 70 C-terminal residues are conserved. As has been shown for yeast TOA1, the internal region serves mainly as a spacer region, contributing little to TOA1 function (14). Sequence alignments of the N- and C-terminal regions showed that some of the most conserved amino acid residues at both termini are present in the T. brucei sequence, supporting the notion that this protein is the orthologue of the large TFIIA subunit (Fig. 5B). Although it will require a functional analysis for a final assignment, we have tentatively named this protein TbTFIIA-1. TRF4, SNAPc, and TFIIA form a stable protein complex. The protein profile of the TRF4-PTP purification raised the possibility that the six major proteins form a single complex. Alternatively, TRF4 may form independent complexes with TFIIA and SNAPc. To discriminate between these two possibilities, we C-terminally PTP-tagged TbSNAP2, TbSNAP50, and TbTFIIA-2 in separate cell lines and conducted PTP purifications. In all three cases, the same six major proteins were purified, strongly indicating that they form a single complex (Fig. 6A). Moreover, the purification pattern did not change when the potassium chloride concentration of the extract was increased to 1 M prior to purification, demonstrating that the tripartite complex is highly salt resistant (data not shown). Due to its larger size, the tagged protein in each purification shifted up in denaturing gel electrophoresis, again confirming that we identified and tagged the correct proteins (Fig. 6A, see arrows).
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To further establish the existence of a tripartite TRF4/SNAPc/TFIIA complex, we analyzed cosedimentation of the subunits in sucrose gradients. As expected, the six major proteins of the TbSNAP2-P eluate cosedimented in stoichiometric amounts in fractions 12 through 14 (Fig. 6B). A complex composed of six monomers has a predicted molecular mass of 230.6 kDa. In accordance with this size, the complex sedimented faster than the 150-kDa large IgG control. Furthermore, increasing the potassium chloride concentration of the gradient to 400 mM did not change the sedimentation properties of the complex, verifying its salt stability (data not shown). Fraction 10 contained detectable amounts of only TbSNAP50 and TbSNAP2, indicating that the TbSNAP2-P final eluate contained both the tripartite complex and a separate SNAPc. Taken together, we concluded that in T. brucei TRF4, SNAPc, and TFIIA form a stable tripartite complex.
TRF4/SNAPc/TFIIA subunits specifically bind to the SL RNA gene USE. Previously we employed a promoter pull-down assay to show that TbSNAP50 binds specifically to the SL RNA gene USE (32, 33). We therefore explored the possibility that the other components of the TRF4/SNAPc/TFIIA complex bind the SL RNA gene promoter with the same specificity. Thus far, we had generated cell lines expressing PTP-tagged TRF4, SNAP2, and TFIIA-2. For the specific detection of the other two components, we generated a cell line which expressed TFIIA-1 with an HA tag fused to its N terminus and raised a polyclonal antiserum against SNAP3. The pull-down assay was carried out with transcription extract of the various cell lines and with linear, biotinylated DNAs which were immobilized on streptavidin beads. The SL RNA gene promoter fragment extended from position –126 to –18 relative to the transcription initiation site covering the USE but not the putative initiator element (Fig. 7A, SL RNA gene fragment –126/–18). As a negative control, we used a DNA fragment covering the GPEET promoter from position –246 to –3 because this DNA did not bind TbSNAP50 in our previous studies (32, 33). Correspondingly, the GPEET –246/–3 pull-down did not reveal binding of any of the TRF4/SNAPc/TFIIA subunits above background levels (Fig. 7B). In contrast, all subunits bound to the SL RNA gene fragment –126/–18. Subunit binding was independent of the PTP tag because a PTP-tagged version of the largest subunit of RNA polymerase I did not bind to SL RNA gene fragment –126/–18. Importantly, subunit binding to SL RNA gene fragment –126/–18 was sequence specific because mutation of USE1, the first sequence block of the bipartite USE, abolished subunit binding in all cases. Mutation of USE2 reduced subunit binding but did not abolish it, indicating that USE1 is the primary site for stable DNA-protein interaction. The exception was HA-tagged TFIIA-1, which in contrast to the other subunits, was unable to bind to the USE2 mutation, a phenotype which has likely been caused by the tag (Fig. 7B, panel HA-TFIIA-1). For comparison, extract corresponding to 20% of the input material was loaded. Although the binding efficiencies of the tagged subunits to SL RNA gene fragment –126/–18 were variable, binding of TFIIA-2-P was especially low, indicating that the tag partially impaired protein function here as well (panel TFIIA-2-P). Finally, we analyzed whether the purified protein complex was capable of specific interaction with the SL RNA gene USE. The pull-down was carried out with SNAP2-P eluate. While purified protein clearly bound to the wild-type USE, mutation of either USE1 or USE2 abolished the interaction, demonstrating the sequence specificity of protein binding. The latter finding was in contrast to the corresponding experiment carried out with extract, in which the same complex was able to bind to the DNA carrying the USE2 mutation (compare panel SNAP2-PTP with panel SNAP2-P). The discrepancy suggests that the extract harbored proteins which stabilized the DNA-protein interaction but which did not copurify with TbSNAP2-P.
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Purified TRF4/SNAPc/TFIIA directs SL RNA gene transcription in vitro. In a final step, we analyzed the function of TRF4/SNAPc/TFIIA in SL RNA gene transcription. We first generated the clonal cell line TbA3, which exclusively expressed TbSNAP2-PTP (Fig. 8A). Importantly, the fact that this cell line grew normally proved that the PTP tag does not inherently interfere with protein function. Moreover, it allowed us to deplete TbSNAP2 from the extract through IgG affinity chromatography (Fig. 8B). Since the depletion step was carried out exactly in the same way as the first PTP purification step, it can be inferred from the protein profile of the TbSNAP2-PTP purification (Fig. 6A, lane 3) that depletion included the TRF4/SNAPc/TFIIA complex. To analyze the effect of complex depletion on SL RNA gene transcription, we carried out in vitro reactions in which we cotranscribed the template constructs GPEET-trm and SLins19. SLins19 contains a complete SL RNA gene tagged with an unrelated oligonucleotide sequence inside its coding region (7). The construct GPEET-trm is similarly tagged and served as a control template because it harbors the GPEET procyclin (GPEET) promoter, which recruits RNA polymerase I (6, 19), and because, as shown in Fig. 7, this promoter does not detectably bind any of the TRF4/SNAPc/TFIIA subunits. The sequence tags in both constructs allow specific detection of the corresponding transcripts by primer extension assays (Fig. 8C). IgG affinity chromatography was carried out by rotating the extract for 2 h at 4°C. When we mock treated transcription extract, we saw that the conditions of IgG affinity chromatography reduced the transcription signals only slightly and that the production of a transcriptionally competent extract of the IgG flowthrough was feasible (compare lanes 1 and 2). Indeed, GPEET transcription was as efficient in TRF4/SNAPc/TFIIA-depleted extract as in mock-treated extract, confirming that the chromatography step did not interfere with transcriptional activity. In contrast to GPEET transcription, SL RNA gene transcription was completely abolished in the depleted extract, indicating that TRF4/SNAPc/TFIIA depletion was causing this effect (compare lanes 2 and 3). To prove that this is the case, we reconstituted SL RNA gene transcription by adding final eluates of the PTP purifications. It is important to note here that we were unable to reconstitute SL RNA gene transcription with EGTA-derived eluates even after dialysis against transcription buffer, indicating that depletion of divalent cations irreversibly destroyed the TRF4/SNAPc/TFIIA transcription function (data not shown). However, ProtC-tagged protein can also be eluted in the presence of ProtC peptide. Therefore, we synthesized this peptide and eluted ProtC-tagged proteins from anti-ProtC matrix in the presence of 0.5 mg/ml peptide. Peptide elution was not as efficient as EGTA elution but resulted in protein concentrations comparable to those in the transcription extract (data not shown). Since transcription reaction mixtures contained 8 µl of extract, addition of 8 µl of peptide eluate approximately restored the original SNAP2 concentration. Accordingly, the SL RNA gene transcription activity was completely reconstituted by the addition of 8 µl of TbSNAP2-P final eluate (Fig. 8C, lanes 5 to 7). Similarly, the TRF4-P eluate was competent for full reconstitution (lane 9). It can be excluded that epitope tagging caused nonspecific activation of SL RNA gene transcription in these assays because, in a control reaction, addition of PTP-purified RNA polymerase I had no effect (lane 4). SL RNA gene transcription was also not reconstituted by the TbSNAP50-P eluate and incompletely restored by the TbTFIIA-2-P eluate, indicating that C-terminal tagging of these proteins interfered with transcriptional function (lanes 8 and 10). Our unsuccessful attempts to establish cell lines exclusively expressing TbSNAP50-PTP (data not shown) are in accordance with the former finding, whereas the latter result is in accordance with the inefficient binding phenotype of TFIIA-2-PTP in the promoter pull-down assays (Fig. 7).
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DISCUSSION |
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Thus far, our analysis has shown that SL RNA gene transcription depends on highly divergent orthologues of human factors which are essential for class II snRNA gene transcription. Since the basal transcription factors TFIIB, TFIIF, and TFIIE are also essential for the latter (15), it is possible that trypanosomatids harbor orthologues of these factors for SL RNA gene transcription. In analyzing proteins which copurified with TRF4-P, we identified TbBRF1. BRF1 is a subunit of TFIIIB and belongs to the highly conserved TFIIB family of proteins which extends to archaea (27). Interestingly, while BRF1 orthologues can be found in the databases of other trypanosomatids, we were unable to identify an orthologue for TFIIB (data not shown). This is surprising because TFIIB is a key molecule in RNA polymerase II recruitment to a promoter and binds the enzyme directly (26). Similarly, TFIIF and TFIIE orthologues have not been identified in trypanosomatid genome databases. This apparent lack of basal transcription factors may be explained by the polycistronic mode which trypanosomatids use to transcribe their protein-coding genes (reviewed in references 1 and 4). It is not known how RNA polymerase II is recruited to these genes, and no class II promoter with a clearly defined transcription initiation site has been characterized for these genes thus far. Hence, it is possible that trypanosomatids have invented a mode of transcription initiation which is independent of most basal transcription factors. On the other hand, characterization of the TRF4/SNAPc/TFIIA subunits in this study suggests that the SL RNA gene promoter forms a class II preinitiation complex comparable to that of other eukaryotes. It may very well be that it is the only such promoter in trypanosomatids. If this is true, the basal factors of trypanosomatids may have diverged much faster than in other organisms because the constraint on function on many different promoters did not exist. Sequence divergence may have progressed to an extent that hampers factor identification by database mining. Hence, biochemical purification will be required to identify other components of the SL RNA gene preinitiation complex. It is possible that some of these factors copurified with TFIIA-2-PTP as minor bands (Fig. 6A, lane 6). Support for this notion comes from the finding that TFIIE and TFIIF directly interact with TFIIA in the human system (17).
In contrast to these basal transcription factors, SNAPc had been characterized only in higher eukaryotes and was not found in S. cerevisiae and S. pombe (12), indicating that it originated late in eukaryotic evolution. The identification of SNAPs in trypanosomatids, however, showed that this transcription factor has a very early evolutionary origin.
Finally, the finding that SL RNA gene transcription in trypanosomatids depends on proteins with extremely divergent sequences raises the possibility that this has structural consequences which can be exploited as antiparasitic targets. Our study represents a critical step in this direction.
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ACKNOWLEDGMENTS |
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We thank Jens Brandenburg for his advice on keeping purified proteins in solution, Laurie Lomask for excellent technical assistance, and Mary Ann Gawinowicz (Protein Core Facility, Columbia University) for superb mass spectrometric analysis.
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FOOTNOTES |
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Supplemental material for this article may be found at http://mcb.asm.org/.
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N-RPA2). In small-scale reactions (100 µl), TbSNAP50-PTP was precipitated by IgG beads and released by TEV protease cleavage. Comparable amounts of input material (INP), of supernatant (S), and of released proteins from the precipitate (P) were analyzed by immunoblotting. On the same blot, TbSNAP50-PTP was detected with anti-ProtC antibody, HA-tagged proteins with anti-HA epitope antibody, and as a control, TbRPA1 with a specific polyclonal antiserum. 
