In-vitro competition analysis of procyclin gene and variant surface glycoprotein gene expression site transcription in Trypanosoma brucei

Molecular & Biochemical Parasitology 113 (2001) 55 – 65 www.parasitology-online.com.

In-vitro competition analysis of procyclin gene and variant surface glycoprotein gene expression site transcription in Trypanosoma brucei Gabriele Laufer, Arthur Gu¨nzl * Zoologisches Institut der Uni6ersita¨t Tu¨bingen, Abteilung Zellbiologie, Auf der Morgenstelle 28, D-72076 Tu¨bingen, Germany Received 27 July 2000; received in revised form 7 November 2000; accepted 30 November 2000

Abstract In Trypanosoma brucei, a-amanitin-resistant transcription characteristic of RNA polymerase I is initiated at ribosomal RNA gene (RRNA), procyclin gene (GPEET or EP1 ), and variant surface glycoprotein gene expression site (VSG ES) promoters. The three promoter types do not share obvious sequence homologies, but contain a proximal domain I and a distal domain II within 80 bp upstream of the transcription initiation site. RRNA, GPEET and EP1, but not the VSG ES promoter, require additional upstream sequences for full activity. In the present study, we competed in-vitro transcription of circular template DNA with linear DNA fragments to identify promoter domains responsible for binding and sequestering essential trans-acting transcription factors. For the GPEET promoter, we found that domain III, located between positions −141 and − 92, was most important for the DNA fragment to exert a transcription competition effect, whereas domain I, the only element absolutely required for transcription, was not. Moreover, insertions between promoter domains II and III reduced both transcription from the GPEET promoter and competition with the GPEET promoter fragment, suggesting that these two domains cooperate in the formation of a stable DNA–protein complex. Taken together, these results indicate a promoter structure very similar to that of the Saccharomyces cere6isiae RRNA promoter. In contrast, VSG ES promoter analysis showed that domains I and II are both necessary and sufficient to compete transcription. Despite this structural difference, our analysis provide evidence that GPEET and VSG ES promoters interact with a common factor that is also important for RRNA promoter transcription. © 2001 Elsevier Science B.V. All rights reserved. Keywords: In-vitro transcription; Procyclin; Promoter structure; RNA polymerase I; Trypanosoma brucei; Variant surface glycoprotein

1. Introduction In eukaryotes, RNA polymerase I transcribes exclusively the large ribosomal RNA gene unit (RRNA). mRNA synthesis is linked to RNA polymerase II because essential mRNA capping occurs co-transcriptionally, with the capping enzyme interacting with the carboxy-terminal domain of the RNA polymerase II largest subunit [1–3]. The protist parasite Trypanosoma * Corresponding author. Tel.: +49-7071-2978862; fax: +49-7071294634. E-mail address: [email protected] (A. Gu¨nzl).

brucei appears to be an exception to this rule. Nuclear run-on assays have revealed that, in addition to RRNA transcription, transcription of procyclin genes and of variant surface glycoprotein gene expression sites is resistant to a-amanitin [4–6], a hallmark of RNA polymerase I-mediated transcription. Production of functional mRNA by RRNA promoter-directed, RNA polymerase I-mediated gene expression has been demonstrated in this organism [7,8] and is possible because mRNA capping is uncoupled from transcription. In T. brucei, mRNAs are capped post-transcriptionally by trans splicing, a process in which the same capped 39 nt-long Spliced Leader (SL) sequence derived

0166-6851/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 6 8 5 1 ( 0 0 ) 0 0 3 8 0 - 7

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from the SL RNA is spliced onto the 5% end of each mRNA molecule. Variant surface glycoprotein gene expression sites (VSG ES) are polycistronic transcription units that are located near telomeres and comprise a single VSG gene and several expression site associated genes. There are about 20 VSG ES per cell, and known sequences of VSG ES promoters are nearly identical [9 – 11]. Procyclin genes are found in two chromosomeinternal, polycistronic gene loci, previously referred to as PARP A and PARP B, but recently renamed GPEET/PAG3 and EP1 /PAG1 -2, respectively [12]. Appropriately and for simplicity, we call the promoters of these transcription units by the name of the first gene of each locus, namely the GPEET promoter (previously PARP A promoter) and EP1 promoter (previously PARP B promoter). Sequences of both promoters exhibit a high degree of homology [6,13]. The structures of the VSG ES promoter [14,15], of GPEET and EP1 promoters [16,17], and of the RRNA promoter [18 –20] have been characterized in detail by transient reporter gene expression assays. All three promoter types possess two essential elements within 80 bp upstream of the transcription initiation site, but do not share clearly conserved sequence motifs. In the VSG ES promoter, the two elements suffice for full promoter activity, whereas procyclin and RRNA promoters require an upstream control region for maximum transcription efficiency. DNA – protein interactions at these promoters have been analyzed by electrophoretic mobility shift assays, and specific band shifts of single- and double-stranded probes have been reported for all three promoter types [14,21 – 24]. Furthermore, Vanhamme et al. [14] provided evidence for a protein of 40 kDa that binds specifically to singlestranded probes covering the distal or the proximal element of the VSG ES promoter, and competition of this band-shift suggested that the binding activity is shared with the GPEET and the RRNA promoter. Recently, we have developed a homologous, cell-free system for T. brucei in which a-amanitin-resistant transcription was efficiently and correctly initiated at RRNA, GPEET, and VSG ES promoters [25]. In the present study, we employed transcription competition using linear promoter fragments as competitors to determine the function of individual promoter domains in stable binding of essential transcription factors. This analysis revealed that, in the GPEET promoter and the VSG ES promoter, different domains are important for the ability of the competitor fragment to sequester an essential factor. Interestingly, our analysis indicated that RRNA, GPEET, and VSG ES promoters recruit at least one common trans-activating transcription factor.

2. Methods

2.1. DNA oligonucleotides, plasmid construction, and generation of competitor DNA fragments The following oligonucleotides were used as transcription competitors or in primer extension assays: ssGPEET − 85/ − 46, 5%-TTGTCCATTTTGTGGCA GTGATGGGGTTGTTTTATGCTATT-3%, ssVSG − 84/ − 45, 5%-TTAACCGTCTAAAAGAATCATATC CCTATTACCACACCAATT-3%, ssVSG − 21/ −50, 5%-CTGTAATAACATCCCCTGTAATATAAATTG3%, Tag – PE [25], and SLtag [26]. Template DNAs GPEET-trm (previously named PARP-trm), Rib-trm, VSG-trm, and SLins19 have been described previously in detail [25,26]. Constructs GPEET/ − 162, GPEET/doIII, GPEET/ − 85ins5, and GPEET/ −85ins11 are derivatives of GPEET-trm. The constructs were generated by replacing the KpnI/ HindIII fragment of GPEET-trm, comprising GPEET promoter positions − 246 to + 120 relative to the transcription initiation site, with DNA fragments which carried site-directed mutations and were made by overlap extension using the polymerase chain reaction (PCR; [27]). In GPEET/ − 162, the GPEET promoter region from position − 246 to position −163 was deleted. In construct GPEET/doIII, the sequence between − 141 and − 92 was replaced by the unrelated sequence 5%-AAATACTGCATTTGAGAGTT TCTCTCCATTGCAAAGTCCTCCCTCTCTTC-3%. GPEET/ − 85ins5 and GPEET/ −85ins11 carry at position − 85 the insertions 5%-CGCGT-3% and 5%CGCGTAGGCCT-3%, respectively. Note, that throughout this article, GPEET and EP1 promoter positions are specified in relation to the transcription initiation site mapped in vitro on the GPEET promoter [25]. Linear competitor fragments were produced by standard PCR using the High Fidelity Expand System (Roche), separated by agarose gel electrophoresis, and purified from gel slices with the QIAquick gel extraction kit (Qiagen) according to the manufacturer’s protocol. In general, each competitor fragment is characterized by its name, which refers to the promoter from which it is derived, e.g. GPEET, RRNA, and VSG, and numbers that denote the positions relative to the transcription initiation site of the 5% and 3%-terminal nucleotides. The competitor fragment GPEET − 162/ − 44ext carried upstream and downstream of the GPEET promoter sequence the unrelated sequences 5%-CCAGGAGGCTCCGCCGGAC TAGAGGTC-3% and 5%-GTGCGAAAAGGACTGAG TAAAATACTGC-3%, respectively. The VSG − 45/ − 3ext competitor fragment was obtained by cloning the VSG − 45/ − 3 promoter sequence into the SmaI site of pUC18 and by PCR amplification of the corre-

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sponding plasmid with M13 Reverse Primer and M13 Universal Primer (Pharmacia). The control competitor fragment Sltelo comprised 222 bp of the Stylonychia lemnae telomerase RNA gene [28].

2.2. Trypanosome culture and extract preparation Cultivation of procyclic forms of T. brucei brucei strain 427 and preparation of cell-free extracts competent in a-amanitin-resistant transcription were carried out as described previously [25].

2.3. In-6itro transcription and RNA analysis In-vitro transcription, RNA preparation, and detection of specific transcripts by primer extension analysis were conducted essentially as described previously [25]. Briefly, transcription reactions were carried out in a volume of 40 ml for 60 min at 28°C, and contained 8 ml of cell extract, 20 mM potassium L-glutamate, 20 mM KCl, 3 mM MgCl2, 20 mM Hepes-KOH, pH 7.7, 0.5 mM ATP, 0.5 mM GTP, 0.5 mM CTP, 0.5 mM UTP, 20 mM creatine phosphate, 0.48 mg ml − 1 of creatine kinase, 2.5% polyethylene glycol, 0.2 mM EDTA, 0.5 mM EGTA, 4.25 mM dithiothreitol, 10 mg ml − 1 of leupeptin, and template DNA. GPEET promoter constructs or pUC18 vector DNA were added to a concentration of 40 mg ml − 1 and co-transcribed with 5 mg ml − 1 of the control template SLins19. In transcription competition experiments, competitor DNA, cell-free extract, and reaction components were preincubated for 15 min on ice before transcription was started by adding template DNA and nucleoside triphosphates. Competition experiments contained 20 mg ml − 1 of GPEET-trm, Rib-trm, or VSG-trm template DNA, 7.5 mg ml − 1 of SLins19, and 12.5 mg ml − 1 of a mix of linear competitor and pUC18 vector DNA. It should be noted that the competitive effect of the RRNA promoter fragment on GPEET-trm, Rib-trm, or VSG-trm transcription was the same when conducted in the presence or absence of the SLins19 template (data not shown). After transcription reactions, RNA was prepared by the single-step method of Chomczynski and Sacchi [29], and in-vitro-synthesized transcripts were specifically detected by primer extension using 32P-end-labeled oligonucleotides. Transcription signals were separated on 6% polyacrylamide – 50% urea gels, visualized by autoradiography, and quantified by densitometry using the E.A.S.Y Win32 imaging system (Herolab). 3. Results

3.1. GPEET, RRNA and VSG ES promoter fragments bind a common and essential transcription factor We employed our in-vitro system for a-amanitin-resis-

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tant transcription in T. brucei [25] to characterize DNA – protein interactions at the GPEET promoter, the RRNA promoter, and a VSG ES promoter by transcription competition experiments using linear promoter fragments as competitors. The aim of this approach was to determine the ability of promoter fragments to bind and sequester essential transcription factors, thereby competing template DNA transcription. In transcription competition assays, we preincubated promoter fragments with cell extract for 15 min and subsequently started transcription reactions by adding template DNA and nucleoside triphosphates. As template DNA, we used the three constructs GPEET-trm, Rib-trm, and VSG-trm containing the GPEET, the RRNA, and a VSG ES promoter, respectively [25]. All three constructs were marked by the same, unrelated 19 bp long sequence, enabling specific detection of in-vitro-synthesized RNA by primer extension with the 5% end-labeled oligonucleotide Tag – PE. Transcription signals were quantified by densitometry. and for normalization of transcription efficiencies, we co-transcribed construct SLins19, which harbors a tagged version of the SL RNA gene and which is not transcribed by RNA polymerase I. SLins19 transcripts were specifically detected by extension of the 5% end-labeled primer SLtag. Transcription signals and competitive effects varied to some extent depending on the extract preparation used. Therefore, each experiment was repeated three times independently. In a first set of experiments, transcription of either GPEET-trm (Fig. 1A), Rib-trm (Fig. 1B), or VSG-trm (Fig. 1C) was competed with a three- or 10-fold molar excess of DNA fragments GPEET − 246/ −3 (fragment comprises the GPEET promoter sequence from position − 246 to − 3 relative to the transcription start site, lanes 3 and 4), rDNA − 257/ − 3 (lanes 5 and 6), and VSG − 84/ − 3 (lanes 7 and 8). These three competitor fragments encompassed all elements essential for maximal transcription from the respective promoters. Control reactions contained no competitor DNA (lane 1) or a 10-fold molar excess of an unrelated, 222 bp long DNA fragment (lane 2). In comparison with the non-specific competitor, the GPEET − 246/ −3 fragment was able to decrease transcription of all three gene constructs considerably (compare lanes 3 and 4 with lane 2 of Fig. 1A–C), demonstrating that it was able to sequester a common trans-activating factor. However, the competition effect on transcription of the three constructs differed. Whereas a threefold molar excess of GPEET − 246/ − 3 strongly inhibited GPEET-trm and Rib-trm transcription, a 10-fold molar excess of this fragment was necessary to obtain a clear inhibitory effect on VSG-trm transcription. Competition with the RRNA promoter fragment rDNA − 257/ − 3 was similar in that it was able to compete transcription of the three constructs (compare Fig. 1A–C, lanes 5 and 6 with lane 2). In comparison to the GPEET − 246/ −3 fragment, however, rDNA − 257/ − 3 competed GPEET-trm and Rib-

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Fig. 1. Competition of GPEET-trm, Rib-trm, and VSG-trm transcription in vitro with full-length promoter fragments. Cell extract was preincubated without competitor (no comp), with a 10-fold (10×) molar excess of non-specific competitor fragment Sltelo (nonspec comp), or with a three- or 10-fold molar excess of specific competitor fragments GPEET − 246/− 3, rDNA − 257/− 3, and VSG −84/−3. Subsequently, templates GPEET-trm (A), Rib-trm (B), or VSG-trm (C) were added and transcribed in vitro together with the control template SLins19. GPEET-trm (GPEET), Rib-trm (Rib), and VSG-trm (VSG) transcripts were detected by primer extension of total RNA prepared from transcription reactions with oligonucleotides Tag – PE, which is complementary to a tag sequence present in all three constructs. Oligonucleotide SLtag was used to detect SLins19 transcripts (SL). The primer extension products were separated on 6% polyacrylamide/50% urea gels and visualized by autoradiography. Arrows on the left indicate marker (M, MspI-digested pBR322) fragment lengths in bp and arrows on the right point to expected positions of transcription signals. Arrows labeled VSG* mark VSG-trm transcription signals generated by aberrant transcription initiation upstream of the correct start site.

the limiting factor with a higher affinity (compare Fig. 1A and B, lanes 3 and 4 with lanes 5 and 6). Unexpectedly, the rDNA − 257/ − 3 fragment competed transcription of the SL RNA control gene, preventing normalization of transcription signals in these reactions (lanes 5 and 6). Interestingly, this finding suggests that RRNA and SL RNA gene promoters, which both recruit different RNA polymerases, share a common transcription factor. This factor does not bind to the GPEET and VSG ES promoter fragments because they did not detectably interfere with SL RNA gene transcription (Fig. 1, transcription signals marked SL, and data not shown). Finally, fragment VSG − 84/ −3 efficiently competed GPEET-trm and Rib-trm transcription (Fig. 1A and B, compare lanes 2 with lanes 7 and 8). A noteworthy effect was seen when the VSG ES promoter fragment competed its own transcription in VSG-trm reactions. Strong signals appeared above the transcription signal of correct length (Fig. 1C, lanes 7 and 8). These signals could be reproduced in the presence of 200 mg ml − 1 of a-amanitin (data not shown) and were most likely generated by transcription initiation upstream of the correct start site. They were not seen when VSG-trm transcription was competed with GPEET and RRNA promoter fragments. This result suggests that a VSG ES-specific factor determines correct transcription initiation at the VSG ES promoter. Furthermore, while the correct VSG-trm transcription signal was reduced in intensity in these reactions, the combined signal strength of correct and aberrant signals was up to five times stronger than in the control reactions (Fig. 1C, compare lanes 2 and 7). Thus, it appears that the VSG −84/ − 3 fragment removed an inhibitory effect of VSG-trm transcription. In sum, we conclude that the GPEET, RRNA and VSG ES promoter fragments were able to stably bind and sequester one or more trans-activating transcription factors. Since each promoter fragment competed transcription of all three templates, we conclude that a common factor interacted with the three promoter fragments. Although our competitor fragments did not include the transcription start site, this common factor may be the RNA polymerase itself because, in other systems, it was shown that binding of RNA polymerase I to the pre-initiation complex is directed exclusively by interactions with a transcription factor and is sequenceindependent [30].

3.2. In-6itro characterization of GPEET promoter domains Next, we wanted to assess the importance of individual promoter elements for stable binding of essential transcription factors. We first focused on the GPEET

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Fig. 2. In-vitro transcription analysis of mutated GPEET promoter constructs. (A) Schematic representation (to scale) of a procyclin gene promoter according to the combined results of Sherman et al. [16] with respect to the GPEET promoter and of Brown et al. [17] with respect to the EP1 promoter. Promoter domains I–IV are represented by black rectangles, and the positions of each domain are indicated relative to the transcription initiation site (flag). Minimal transcription efficiencies observed with mutations inside each domain in vivo [16,17], and in vitro ([25], this study) are given as a percentage of wild-type transcription. n.d., not detectable. (B) Constructs GPEETtrm, GPEET/− 162 ( −162), GPEET/doIII (doIII), GPEET/− 85ins5 ( −85ins5), and GPEET/− 85ins11 ( −85ins11) were transcribed in vitro together with the control template SLins19. As a negative control, vector DNA was transcribed (vector). Transcription signals were obtained by primer extension with 5% end-labeled oligonucleotides Tag – PE and SLtag, which are complementary to GPEET-trm and the SLins19 RNAs, respectively. Expected positions of GPEET-trm (GPEET) and SLins19 (SL) transcription signals are indicated by arrows on the right and sizes of marker (M) fragments in bp by arrows on the left. Transcription signals were quantified by densitometry, and below each lane, the average signal strength and standard deviation from three independent experiments are expressed as a percentage of wild-type GPEET-trm transcription.

promoter because its structure is known in detail, and transcription from this promoter is very strong in our procyclic cell extract. To do this, we first determined the importance of individual promoter domains for in-vitro transcription by mutational analysis. According to results obtained in vivo by Sherman et al. [16] on the GPEET promoter and Brown et al. [17] on the nearly identical EP1 promoter, the region between positions −246 and −13 is required for full transcriptional activity, and two distinct domains are located between positions −13 and −40 (domain I) and between − 57 and − 72 (domain II; see Fig. 2A). In the same studies

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[16,17], linker scanner analyses in the upstream control region produced equivocal results. Distinct effects were observed between positions − 90 and − 143 (domain III) and between positions − 207 and − 230 (domain IV; see Fig. 2A). Previously, we have demonstrated that, in close accordance with these results, mutation of domain I abolished GPEET promoter transcription in vitro, and mutation of domain II reduced the transcription signal strength approximately eightfold [25]. In the same study, a 5% deletion to position − 84 reduced the transcription signal to approximately the same level, demonstrating that the upstream region is important for maximal transcription efficiency in vitro. Here, we analyzed the upstream region further; several gene constructs were generated and tested in co-transcription assays with the control template SLins19 (Fig. 2B). In construct GPEET-trm/ − 162, the 5% terminal part of the GPEET promoter extending to position − 162 was deleted. In comparison to the undeleted promoter, the transcription signal of GPEET-trm/ − 162 was reduced by only 8%, demonstrating that domain IV contributes little to transcription efficiency in vitro (Fig. 2B, compare lanes 2 and 3). A promoter domain located at this position (− 222 to −207; see Fig. 2A), and important for transcription in vivo but not in vitro, is reminiscent of the proximal terminator, an RRNA promoter element found in eukaryotes as diverse as yeast and mammals (reviewed in Refs. [31,32]). This element was shown to be particularly important in vivo, because it is involved in remodeling of a repressive chromatin structure ([31] and references therein). Thus, the GPEET domain IV may represent the GPEET proximal terminator. The insignificance of GPEET domain IV suggested that domain III may have a major part in GPEET promoter function. In accordance with this hypothesis, replacement of the 50 bp long region from position − 141 to − 92 with an unrelated sequence decreased transcription efficiency of the corresponding construct GPEET-trm/doIII by 67% (Fig. 2B, lane 4). Sequences upstream of position − 141 were less important because mutating the sequence from position −141 to −170 reduced the transcription signal strength by only 12% (data not shown). The importance of domain III for GPEET promoter transcription was confirmed by increasing the distance between domains II and III. Gene constructs GPEET-trm/ − 85ins5 and GPEETtrm/ − 85ins11 carried 5 and 11 bp long insertions at position − 85, respectively, and produced transcription signals that both had a strength of only 11% of the wild-type level (Fig. 2B, lanes 5 and 6). This strong decline in transcription efficiency is in agreement with results previously obtained in vivo [16,17] and shows that the exact position of domain III is crucial for the GPEET promoter to function efficiently.

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3.3. GPEET promoter domain III is essential for stable protein binding To analyze which GPEET promoter domain is important for stable protein binding, we first investigated whether DNA fragments comprising individual promoter domains were able to sequester a trans-activating factor of GPEET-trm transcription. However, even a 30-fold molar excess of fragments GPEET − 44/ − 3, GPEET − 84/ −46, or GPEET −162/ − 86 containing promoter domains I, II, and III, respectively, did not exert a competitive effect on GPEET-trm transcription (data not shown; see Table 1). Since Brown and Van der Ploeg [22] had previously reported a sequence-specific band shift with a single-stranded sense oligonucleotide probe encoding the EP1 promoter sequence from position −79 to position − 50, we analyzed whether the single-stranded oligonucleotide ssGPEET −84/ − 46 was able to compete GPEET-trm transcription, but no competitive effect even at 30-fold molar excess was detected (data not shown; see Table 1). Hence, we concluded that DNA fragments must have a combination of promoter domains to be able to sequester an

essential transcription factor. To characterize a minimal competitor fragment, we progressively deleted the GPEET − 246/ − 3 fragment, first from its 5% end (Fig. 3). Deletion of domain IV in fragment GPEET −162/ −3 reduced the competitive effect only slightly in comparison to the full size fragment (Fig. 3B; compare lanes 3 and 4 with lanes 5 and 6; see Table 1). In contrast, deleting the fragment to position − 84 and removing domain III resulted in a complete loss of transcription competition (Fig. 3B, lanes 7 and 8), demonstrating that domain III is of crucial importance for the competitor fragment’s ability to sequester a trans-activating factor. In a second set of experiments, the GPEET −246/ −3 competitor fragment was progressively deleted from its 3% end (Fig. 4). Deletion of promoter domain I to position − 44 barely affected the competitive ability of the fragment (Fig. 4B, lanes 5 and 6), demonstrating that domain I contributes little to the formation of a stable DNA –protein complex, though it is the only domain absolutely essential for the generation of transcription signals [16,17,25] and may therefore represent the core promoter. Deleting the fragment further to

Table 1 Summary of transcription competition experimentsa Template

GPEET-trm

Competitor fragment

3×

10×

GPEET −246/−3 GPEET −246/−3 doIII GPEET −246/−3 doII GPEET −246/−3 doI GPEET −246/−3 ins5 GPEET −246/−3 insT 1 GPEET −246/−44 GPEET −246/−84 GPEET −246/−100 GPEET −246/−162 GPEET-162/−3 GPEET −162/−44ext GPEET −162/−84b GPEET −84/−3 GPEET −84/−46b GPEET −44/−3b ssGPEET −84/−46b (sense) rDNA −257/−3c VSG −84/−3 VSG −162/−46b VSG −44/−3extb ssVSG −84/−46b (sense) ssVSG −21/−50b (antisense)

++

++

a

+ − − − +

−

− +

Rib-trm

+ ++ + + ++ + + − ++ + − − − − − + ++

30×

VSG-trm

3×

10×

++

++

30×

3×

10×

−

+

++

30×

+

+

+ + − − − −

− − − −

− − − − + ++

++ ++

− − − −

In-vitro transcription of circular DNA templates GPEET-trm, Rib-trm, or VSG-trm were competed with three-, 10-, or 30-fold molar excess of linear DNA fragments of the corresponding promoters. Transcription signals were quantified by densitometry, normalized with signals from the co-transcribed SLinsl9 template and compared to reactions with non-specific competitors. Competitive effects were determined by a minimum of three independent experiments and are expressed as ++ (greater than 80% reduction of transcription signal), + (50–80%), and − (less than 50%). b Data not shown. c Reactions with the fragment rDNA −257 /−1 were not normalized because it competed transcription of the SLinsl9 control template.

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dance with the deletion analysis, mutation of domain III resulted in the strongest reduction of the competition effect (lane 3). Mutating domain II or increasing the distance between domains II and III moderately reduced the competition effect (lanes 4, 6, and 7), and mutation of domain I, the most important domain for GPEET transcription, hardly affected the DNA fragment’s competition ability (lane 5). Taken together, these data show that GPEET promoter domain III is essential for the transcription competition effect because fragments lacking this domain were unable to compete. However, domain III alone was not sufficient for the effect, suggesting that it must interact with other domains for stable association with essential transcription factors. Accordingly, a competitor fragment comprising domains III and IV competed GPEET-trm transcription. Moreover, the reduced competition ability of GPEET − 246/ − 3

Fig. 3. 5% deletions of the GPEET competitor fragment. (A) Schematic outline (to scale) of the GPEET promoter and the competitor fragments GPEET − 246/− 3, GPEET − 162/− 3, and GPEET − 84/ − 3. Promoter domains I–IV are indicated by black rectangles. (B) In-vitro GPEET-trm transcription was competed with a three- or 10-fold molar excess of 5% deleted GPEET promoter fragments. Control reactions were carried out in the absence of competitor and with a 10-fold molar excess of the non-specific competitor fragment Sltelo (nonspec comp). Arrows mark GPEET-trm transcription signals (GPEET) and signals of the co-transcribed SLins19 template (SL). M, marker (MspI-digested pBR322).

positions −84 or −100, thereby removing domain II, resulted in a strong loss of competition (Fig. 4B, lanes 7–10). Nevertheless, a 10-fold molar excess of these fragments revealed a distinct competitive effect. In contrast, fragment GPEET −246/ −162, which lacks domain III, did not compete (Fig. 4B, lanes 11 and 12), confirming the importance of domain III for stable protein binding. Partial deletion of the competitor fragment may result in non-specific loss of competition because the DNA fragment may still possess the essential sequence binding motif but may be too short at the deleted end to support stable protein binding. Therefore, to verify the importance of individual promoter domains in GPEET-trm transcription competition, we used a 10-fold molar excess of GPEET − 246/ − 3 competitor fragments carrying internal mutations. Mutated GPEET − 246/ −3 competitor fragments were derived from constructs GPEET-trm/doIII (doIII), GPEET-trm/ − 85ins5 (− 84ins5), and GPEET-trm/ − 85ins11 (−84ins11; see Section 2) as well as from PARP-trm/25.35 (doII) and PARP-trm/ 44.38 (doI, [25]). As shown in Fig. 5 and in accor-

Fig. 4. 3% deletions of the GPEET competitor fragment. (A) Schematic outline (to scale) of the GPEET promoter and the competitor fragments GPEET −246/− 3, GPEET − 246/− 44, GPEET − 246/− 84, GPEET-trm −246/−100, and GPEET − 246/− 162. Black rectangles represent promoter domains I to IV. (B) In-vitro GPEETtrm transcription was competed with a three- or 10-fold molar excess of 3% deleted GPEET promoter fragments. Control reactions were carried out in the absence of competitor and with a 10-fold molar excess of the non-specific competitor fragment Sltelo (nonspec comp). Arrows mark GPEET-trm transcription signals (GPEET) and signals of the co-transcribed SLins19 template (SL). M, marker (MspI-digested pBR322).

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3.5. Competition with VSG ES promoter fragments

Fig. 5. Mutation of the GPEET − 246/− 3 competitor fragment. In-vitro transcription competition reactions were carried out with mutated GPEET − 246/− 3 DNA fragments and GPEET-trm and SLins19 template DNAs. The competitor fragment was mutated in domain III (doIII), domain II (doII), or domain I (doI), or carried 5 ( − 85ins5) or 11 bp long ( − 85ins11) insertions at position −85. A control reaction was carried out with the non-specific competitor fragment Sltelo (nonspec comp). Competitor fragments were added in 10-fold molar excess. Arrows point to expected positions of GPEETtrm (GPEET) and SLins19 (SL) transcription signals. M, marker (MspI-digested pBR322).

As determined in vivo, the VSG ES promoter lacks an upstream control region and consists of only two elements, which are similarly positioned relative to the transcription start site as procyclin promoter domains I and II [14,15]. We verified the absence of additional promoter elements by extending the VSG ES promoter of the VSG-trm construct from position − 100 to position − 250 and observed no alteration in in-vitro transcription efficiency (data not shown). Accordingly, the VSG − 84/ − 3 fragment that comprised the whole VSG ES promoter was an efficient transcription competitor (Fig. 1A–C, lanes 7 and 8). The competitive effect of VSG − 84/ − 3 depended on both promoter elements because fragments VSG −162/ − 44 and VSG −44/ − 3ext containing the distal and proximal promoter element, respectively, were unable to compete VSG-trm transcription (data not shown; see Table 1). Furthermore, since specific band-shifts have been reported for single-stranded probes covering the antisense strand of the proximal VSG ES promoter element and the sense strand of the distal element [14], we used the single-stranded oligonucleotides ssVSG − 84/ − 46 and ssVSG − 21/ − 50 to compete VSG-trm transcription, but failed to detect a competitive effect even at a 30-fold molar excess of the oligonucleotides (data not shown; see Table 1). Hence, our data do not support

fragments carrying insertions at position −84 suggested that domain III may also interact with domain II.

3.4. A GPEET promoter fragment containing domains II and III is able to compete GPEET-trm, Rib-trm, and VSG-trm transcription To test this hypothesis, competitor fragment GPEET − 162/ − 44ext was generated, which encompassed domains II and III and carried additional, unrelated nucleotides at either end. As shown in Fig. 6, GPEET − 162/ − 44ext efficiently competed GPEET-trm (lanes 1 and 2) and Rib-trm (lanes 3 and 4) transcription and was a moderate competitor of VSG-trm transcription (lanes 5 and 6; see Table 1). The competitive effect on VSG-trm transcription became more obvious when the competitor concentration was increased (lanes 7 and 8). Hence, domains II and III of the GPEET promoter cooperate in stable binding of a trans-activating transcription factor common to GPEET, RRNA and VSG ES promoters. Although it cannot be excluded that the factor that was sequestered by the competitor fragment was the RNA polymerase itself, this possibility is unlikely because the competitor fragment did not include domain I, the putative core promoter.

Fig. 6. GPEET promoter domains II and III bind a common transactivating transcription factor. In-vitro transcription of templates GPEET-trm, Rib-trm, and VSG-trm were competed with a 10-fold (lanes 1 – 6) or 30-fold (lanes 7 and 8) molar excess of DNA fragments Sltelo (nonspec) and GPEET −162/− 44ext ( −162/− 44). Marker (M) fragment sizes (in bp) are indicated on the left and transcription signals on the right.

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the hypothesis that VSG ES promoter transcription depends on a single-stranded DNA-binding factor. In sum, our results show that the VSG ES promoter fragment’s ability for binding and sequestering a transactivating transcription factor depends on both the proximal and the distal promoter element and differs significantly from complex formation on the GPEET promoter.

4. Discussion We have employed in-vitro transcription competition assays using linear promoter fragments as competitors to determine the regions of GPEET and VSG ES promoters that are able to bind and sequester trans-activating transcription factors in our crude cell extract. We have shown that different elements are required to achieve a competitive effect in these two promoters. Our analysis of the GPEET promoter revealed a structure remarkably similar to that of the Saccharomyces cere6isiae RRNA promoter. Mutational analyses have shown that the yeast RRNA promoter consists of three domains; domain I ranging from position + 8 to −28, domain II from −51 to − 76, and domain III from −91 to −146 relative to the transcription initiation site [33 –35]. Hence, the size and location of these domains are in close accordance with those of the T. brucei GPEET promoter (see Fig. 2A). Furthermore, the yeast RRNA promoter domain I represents the core promoter because it is the only element absolutely required for transcription. Domain I, however, binds very weakly to the core factor, which is the transcription factor interacting with RNA polymerase I and thus, by itself, is not able to form a stable DNA – protein complex and commit a template for transcription [36]. Rather, formation of a stable pre-initiation complex is mediated by yeast promoter domains II and III that constitute a bipartite upstream element and bind the multi-subunit upstream activation factor [37]. Template competition experiments demonstrated that the factor-binding activity of this element resides mainly in domain III [34]. Our results on the GPEET promoter are in accordance with such a promoter structure: GPEET promoter domain I was the only promoter element absolutely essential for transcription in vivo and in vitro [16,17,25] and probably corresponds to the core promoter. As predicted by the yeast RRNA promoter structure, GPEET promoter domain I contributed very little to the competitive effect of the GPEET competitor fragment. Domain I was unable to compete by itself, and its removal from the competitor fragment barely reduced the competition effect, suggesting that domain I interacts only weakly with a trans-activating factor. In contrast, domain III was essential for stable protein binding, and domain II

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contributed significantly to the competition effect. Moreover, insertions between domains II and III resulted in a dramatic loss of GPEET promoter transcription and strongly reduced competition of DNA fragment GPEET −246/ −3, suggesting that both domains cooperate in stable and correct complex formation and function as a bipartite upstream element. Since the VSG ES promoter lacks domain III, domains I and II must be able to stably recruit essential transcription factors by themselves. Accordingly, we observed strong competition with the VSG −84/ −3 fragment (Fig. 1A–C, lanes 7 and 8), whereas the corresponding GPEET − 84/ −3 fragment did not compete (Fig. 3, lanes 7 and 8). Furthermore, our results showed that the competitive ability of the VSG fragment depended on both promoter elements, suggesting their cooperation in forming a stable DNA – protein complex. This finding is in close agreement with the study of Pham et al. [24], who showed by electrophoretic mobility shift assays that specific protein binding to double-stranded VSG ES promoter probes required the integrity of both elements. Taken together, these results indicate that the VSG ES promoter elements are not functionally analogous to the corresponding GPEET domains I and II. The two-domain structure of the VSG ES promoter is reminiscent of RRNA promoters of higher eukaryotes [38], which possess a core element and a single upstream element (reviewed in Ref. [39]). However, it should be noted that the VSG ES domain II is located around position − 60, whereas upstream elements of RRNA promoters of higher eukaryotes are located approximately 90 bp further upstream and are much larger [39]. Therefore, the structure of the VSG ES promoter in a stricter sense does not resemble any known eukaryotic RRNA promoter. Despite the structural difference between GPEET and VSG ES promoters, transcription competition unequivocally showed that both promoters and the RRNA promoter interact with a common trans-activating transcription factor. This factor is specific for transcription initiated at these three promoters because it is not required for SL RNA gene transcription. Hence, it is most likely not a ubiquitous transcription factor such as TATA-binding protein, suggesting that RRNA, procyclin gene and VSG ES transcription is mediated by the same RNA polymerase, namely RNA polymerase I. We were unable to assess whether the limiting factor in transcription competition experiments was the RNA polymerase itself. However, given the fact that promoter fragment GPEET − 162/ − 44ext, comprising promoter domains II and III and lacking the proximal domain I, was able to compete transcription from all three promoters, this indicates that the commonality is a DNA-binding factor. This hypothesis is supported by the finding that an exchange of promoter domains in

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RRNA/GPEET and VSG ES/RRNA hybrid promoters were functional in transient reporter gene assays [14,20,40]. A direct analysis of a shared DNA-binding factor would be possible by an electrophoretic mobility shift assay. We attempted to characterize specific band-shifts with our transcriptionally active extract but failed to reproduce already published band-shifts or to detect any other specific band-shifts. This is most likely due to the complexity of the crude extract that harbors strong non-specific DNA-binding activities (data not shown). A partial purification of the cell extract appears to be necessary for the detection of specific band-shifts. In transcription systems of other organisms, the role of an individual promoter element in the formation of a stable pre-initiation complex has been determined by standard means by template commitment assays. In such an assay, DNA – protein complexes formed on a first DNA template are challenged by the addition of a second DNA template. If the DNA – protein complex of the first template is stable, it is defined as a committed complex. We did not conduct template commitment experiments because our in-vitro transcription system requires circular template DNA. Since a circular template may function as a trap for RNA polymerase I, this, rather than stable binding of transcription factors by the first template, might prevent transcription of the second template. In conclusion, the transcription competition experiments presented here elucidate the role of individual GPEET and VSG ES promoter domains in stable binding of essential transcription factors, and they have provided evidence that one or more common factors interact with these two promoters and the RRNA promoter. Partial purification of the transcription system and the right choice of DNA probe should facilitate a more direct analysis of DNA – protein interactions and eventually lead to purification of transcription factors. Acknowledgements We thank Sigrid Hojak for excellent technical assistance and Robert Paxton for critically reading the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft, grant Gu371/3-1. References [1] Cho EJ, Takagi T, Moore CR, Buratowski S. mRNA capping enzyme is recruited to the transcription complex by phosphorylation of the RNA polymerase II carboxy-terminal domain. Genes Dev 1997;11:3319– 26. [2] McCracken S, Fong N, Rosonina E, Yankulov K, Brothers G, Siderovski D, et al. 5%-Capping enzymes are targeted to premRNA by binding to the phosphorylated carboxy-terminal domain of RNA polymerase II. Genes Dev 1997;11:3306–18.

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