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Cloning and functional characterization of PTRF, a novel protein which induces dissociation of paused ternary transcription complexes

1998; Springer Nature; Volume: 17; Issue: 10 Linguagem: Inglês

10.1093/emboj/17.10.2855

1460-2075

Petr Jansa,

Plant nutrient uptake and metabolism

Article15 May 1998free access Cloning and functional characterization of PTRF, a novel protein which induces dissociation of paused ternary transcription complexes Petr Jansa Petr Jansa Division of Molecular Biology of the Cell II, German Cancer Research Center, 69120 Heidelberg, Germany Search for more papers by this author Stephen W. Mason Stephen W. Mason Present address: Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, M5G1L6 Canada Search for more papers by this author Urs Hoffmann-Rohrer Urs Hoffmann-Rohrer Division of Molecular Biology of the Cell II, German Cancer Research Center, 69120 Heidelberg, Germany Search for more papers by this author Ingrid Grummt Corresponding Author Ingrid Grummt Division of Molecular Biology of the Cell II, German Cancer Research Center, 69120 Heidelberg, Germany Search for more papers by this author Petr Jansa Petr Jansa Division of Molecular Biology of the Cell II, German Cancer Research Center, 69120 Heidelberg, Germany Search for more papers by this author Stephen W. Mason Stephen W. Mason Present address: Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, M5G1L6 Canada Search for more papers by this author Urs Hoffmann-Rohrer Urs Hoffmann-Rohrer Division of Molecular Biology of the Cell II, German Cancer Research Center, 69120 Heidelberg, Germany Search for more papers by this author Ingrid Grummt Corresponding Author Ingrid Grummt Division of Molecular Biology of the Cell II, German Cancer Research Center, 69120 Heidelberg, Germany Search for more papers by this author Author Information Petr Jansa1, Stephen W. Mason2, Urs Hoffmann-Rohrer1 and Ingrid Grummt 1 1Division of Molecular Biology of the Cell II, German Cancer Research Center, 69120 Heidelberg, Germany 2Present address: Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, M5G1L6 Canada *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:2855-2864https://doi.org/10.1093/emboj/17.10.2855 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Termination of transcription by RNA polymerase I (Pol I) is a two-step process which involves pausing of elongating transcription complexes and release of both pre-rRNA and Pol I from the template. In mouse, pausing of elongation complexes is mediated by the transcription termination factor TTF-I bound to the 'Sal box' terminator downstream of the rDNA transcription unit. Dissociation of paused ternary complexes requires a cellular factor, termed PTRF for Pol I and transcript release factor. Here we describe the molecular cloning of a cDNA corresponding to murine PTRF. Recombinant PTRF is capable of dissociating ternary Pol I transcription complexes in vitro as revealed by release of both Pol I and nascent transcripts from the template. Consistent with its function in transcription termination, PTRF interacts with both TTF-I and Pol I. Moreover, we demonstrate specific binding of PTRF to transcripts containing the 3′ end of pre-rRNA. Substitution of 3′-terminal uridylates by guanine residues abolishes PTRF binding and impairs release activity. The results reveal a network of protein–protein and protein–nucleic acid interactions that governs termination of Pol I transcription. Introduction Transcription by all three classes of nuclear RNA polymerases proceeds in distinct steps designated initiation, elongation and termination. Although transcription initiation is a major target for regulation, a growing body of evidence indicates that elongation and 3′ end formation also play important roles in modulating cellular transcriptional activity (reviewed in Manley and Proudfoot, 1994; Shilatifard et al., 1997). Like all other steps in RNA synthesis, formation of the 3′ end of nascent transcripts is a complex process that requires both protein–nucleic acid and protein–protein interactions. Thus, both pausing of the transcription elongation complex and proper 3′ end formation, i.e. dissociation of the ternary transcription complex and 3′-terminal processing of the primary transcript, are mediated by ancillary proteins which recognize specific sequence motifs or structures within DNA or RNA and are capable of communicating with components of the transcription apparatus to terminate transcription. While transcription termination of genes transcribed by RNA polymerase II is still poorly characterized, the mechanism of transcription termination by RNA polymerase I (Pol I) is much better understood. In short, termination of Pol I occurs at specific terminator elements downstream of the pre-rRNA coding region (Grummt et al., 1985; Bartsch et al., 1987). Despite marked differences in the cis-acting elements and trans-acting factors in species as diverse as yeast, Xenopus, human, rat and mouse, the mechanism of Pol I transcription termination in all eukaryotes is probably very similar (reviewed by Reeder and Lang, 1994, 1997; Mason et al., 1998). All characterized Pol I terminator elements function in only one orientation and are recognized by a specific DNA-binding protein that stops elongating Pol I. The terminator protein, i.e. TTF-I in mammals or Reb1p in yeast, presumably contacts the elongating RNA polymerase and mediates the termination reaction. In addition to the binding site for the terminator protein, an upstream element that codes for the last 10–12 nucleotides of pre-rRNA is required for complete termination, e.g. for release of the terminated transcripts and Pol I. In the mouse, termination of Pol I transcription occurs 565 bp downstream of the 28S RNA coding region (Grummt et al., 1985). The 3′ endpoint of the pre-rRNA maps upstream of T1, the first of 10 'Sal box' terminator elements (AGGTCGACCAGA/TT/ANTCCG) which are clustered within several hundred base pairs of the non-transcribed spacer downstream of the 28S rRNA coding region (Grummt et al., 1986). The individual Sal box elements are flanked by long pyrimidine stretches, not uncommon for a eukaryotic terminator. Indeed, a T-rich element upstream of the first terminator (T1) has been demonstrated to be required for both efficient transcript release and 3′-terminal processing (Kuhn and Grummt, 1989; Lang and Reeder, 1995; Mason et al., 1997a). The overall base composition of the upstream element determines the efficiency of transcript release (Kuhn and Grummt, 1989; Lang and Reeder, 1995). The availability of cloned terminator proteins facilitated the establishment of cell-free systems which terminate at the same sites utilized in vivo and thus allowed functional studies concerning the mechanism of transcription termination. These studies revealed that murine Pol I transcription termination requires two cis-acting elements, the Sal box terminator and the T-rich element located upstream of the terminator T1, as well as two trans-acting factors, i.e. TTF-I and a novel activity that dissociates TTF-I-paused transcription complexes (Mason et al., 1997a). This novel activity is now designated PTRF, for Pol I and transcript release factor. PTRF activity was initially identified in partially purified fractions by complementation of a release-deficient cellular Pol I for transcript release. Here we report the cloning and functional characterization of PTRF and demonstrate that the recombinant protein possesses functional properties similar to those of the partially purified cellular factor. Like cellular PTRF, the recombinant factor allows release of nascent Pol I transcripts from ternary transcription complexes that are paused by TTF-I. We demonstrate specific interaction of PTRF with both TTF-I and Pol I, and show that PTRF binds in vitro to transcripts containing the 3′ end of pre-rRNA. Based on these properties of PTRF, a model of transcription termination is presented. Results PTRF mediates dissociation of ternary transcription complexes paused by TTF-I The transcript release assay utilizes the template pCAT-T6-T1 which contains part of the chloramphenicol acetyltransferase (CAT) gene fused to a fragment from the 3′-terminal spacer of mouse rDNA including one Sal box element and flanking sequences (Figure 1A). A 10 nucleotide single-stranded 3′ extension or 'tail' was added to the 5′ end, which facilitates transcription initiation in the absence of auxiliary factors (Kuhn et al., 1990). Attachment of a magnetic bead to the other end of the template allows separation of ternary elongation complexes, which are paused at the terminator and are still attached to the template, from free Pol I and transcripts which are released into the supernatant. Figure 1.Functional properties of cellular PTRF. (A) Diagram showing the structure of the tailed template pCAT-T6-T1 and the mutant pCAT-G6-T1. The positions of the extended 3′ overhang, the CAT fragment (open box) and the 3′-terminal rDNA fragment (thick line) including the T1 terminator element are indicated. The nucleotide sequence of the terminator region is shown below. The 18 bp Sal box terminator element is boxed; bold letters mark the six T residues in the flanking region which are substituted by G residues in the mutant pCAT-G6-T1. Numbers indicate the position of nucleotides with respect to the 3′ end of the 28S RNA coding region. The two vertical arrows mark the position of the primary and 3′-terminally processed transcript whose lengths are 202 and 198 nucleotides, respectively. (B) PTRF facilitates transcript release. Transcript release was assayed on immobilized tailed templates pCAT-T6-T1 (lanes 1–6) or pCAT-G6-T1 (lanes 7–12). Reactions contained 5 μl of Pol I (0.2 U), 20 ng of TTF-I and 0, 3 or 6 μl of cellular PTRF (MonoS fraction, 0.5 ng of PTRF per μl) as indicated. RNA synthesized during a 10 min incubation was fractionated into template-bound (b) and released (r) transcripts. (C) RNA-binding activity of PTRF. 32P-labeled RNA probes containing 20 nucleotides from the 3′-terminus of mouse pre-rRNA (lanes 1–3) were incubated in the absence or presence of cellular PTRF (MonoS fraction) as indicated. In lanes 4–6, a mutant RNA probe was used in which the six U residues were replaced by guanosines. The ribonucleoprotein complexes were separated from unbound RNA by electrophoresis in non-denaturing 5% polyacrylamide gels and visualized by autoradiography. Download figure Download PowerPoint In transcription assays containing Pol I and TTF-I, two closely spaced transcripts are generated, the longer one representing the primary terminated transcript which is converted into the shorter one by a processing reaction that removes four nucleotides from the 3′ end of the nascent transcript (Kuhn and Grummt, 1989; Mason et al., 1997a). In the absence of a PTRF-containing fraction, the majority of transcripts remained bound to the template (Figure 1B, lanes 1 and 2). The proportion of template-bound versus free transcripts in the supernatant changed when increasing amounts of partially purified PTRF (MonoS fraction) were added. At the highest amount of PTRF added, practically all transcripts were found in the supernatant (Figure 1B, lanes 3–6), indicating that ternary complexes were dissociated and the RNA released from the template. Significantly, PTRF function requires DNA sequences upstream of the terminator T1 which affect the efficiency of 3′ end formation (Kuhn and Grummt, 1989; Lang and Reeder, 1995; Mason et al., 1997a). Conversion of the six thymidine residues in the non-template strand (from +566 to +571 with respect to the 3′ end of 28S RNA) into guanosines (pCAT-G6-T1) impairs transcript release (Figure 1B, lanes 7–12). Thus, both PTRF and the T-rich sequence upstream of the terminator T1 are required for transcript release. The importance of both the upstream sequence element and PTRF for dissociation of the ternary elongation complex is consistent with previous data demonstrating that Pol I and transcript release depend on sequences contained in the very 3′ end of pre-rRNA. To examine whether PTRF would bind specifically to the end of the primary rDNA transcript, we used T7 RNA polymerase to synthesize a short RNA which contains the same 3′ end as pre-rRNA and therefore resembles the end of the natural Pol I product. In parallel, a mutant transcript was used where the six U residues were converted into guanosines. Binding of cellular PTRF to both wild-type and mutant RNA was measured in an electrophoretic mobility shift assay. As shown in Figure 1C, incubation of PTRF with the wild-type probe (lanes 1–3) yielded a defined complex which was not formed with the mutant RNA probe (lanes 4–6). This result demonstrates that PTRF binds RNA, and the U-rich element is involved in specific PTRF–RNA interaction. Cloning of PTRF The strategy for cloning the cDNA encoding PTRF was based on preliminary observations indicating that this factor interacts with TTF-I (unpublished results). We therefore performed a yeast two-hybrid screen (Fields and Song, 1989; Gyuris et al., 1993) using TTF-I as a bait. The initial screening of 2×107 clones from a human lung fibroblast WI-38 cDNA library yielded five positive clones, one of which encoded a novel protein which was found to represent an N-terminally truncated version of human PTRF (data not shown). The corresponding full-length murine cDNA was obtained by a PCR-based approach as described in Materials and methods. As will be shown below, this cDNA encodes functional murine PTRF. The deduced amino acid sequence of murine PTRF is shown in Figure 2. The cDNA encompasses a 1176 nucleotide open reading frame (ORF) that predicts a 392 amino acid protein with a molecular mass of 44 kDa. The human and mouse sequences are 94% homologous at the amino acid level and contain two putative nuclear localization signals. Both human and mouse PTRF show 89% homology to a chicken protein that has been reported to DDBJ/EMBL/GenBank and is referred to as a putative leucine zipper protein (Sawada et al., 1996). The marked sequence homology between the human, mouse and chicken cDNA suggests that PTRF is a highly conserved protein. Figure 2.Nucleotide and deduced amino acid sequence of mouse PTRF. The underlined sequences correspond to two putative nuclear localization signals identified by the PROSITE program (Senger et al., 1995). Two clusters of basic amino acids contained in both bipartite NLSs are marked by bold letters. Download figure Download PowerPoint Recombinant PTRF interacts with TTF-I To examine which domain of TTF-I is involved in the interaction with PTRF, a series of N- and C-terminally truncated TTF-I mutants were fused to LexA and transformed into a yeast strain carrying a β-galactosidase reporter and PTRF fused to a transcription activation domain (Figure 3A). In this experiment, the cDNA derived from the initial yeast two-hybrid screen was used which encodes N-terminally truncated human PTRF. The fusion protein containing full-length TTF-I (LexA–TTFp130) reconstitutes the activator required for LacZ expression, resulting in β-galactosidase levels significantly higher than background. Consistent with the N-terminus of TTF-I being dispensable for transcription termination (Evers et al., 1995), no interaction was detected between PTRF and the N-terminal part of TTF-I (TTF1–323). On the other hand, the N-terminal deletion mutant TTFΔN323 which efficiently promotes transcription termination (Evers et al., 1995) produced high levels of β-galactosidase. TTFΔN323-mediated activation of LacZ expression was even higher than that of the full-length protein TTFp130. Thus, in support of previous experiments demonstrating that TTFΔN323 binds to DNA with higher affinity than TTFp130 (Evers et al., 1995; Sander et al., 1996), the C-terminal half of TTF-I, which harbors the domains involved in DNA-binding and termination activity, also mediates the interaction with PTRF. Figure 3.TTF-I interacts with PTRF in vivo and in vitro. (A) The central part of TTF-I interacts with PTRF. Full-length (TTFp130) and defined regions of murine TTF-I were fused in-frame to the LexA DNA-binding domain and tested in the yeast two-hybrid system for their interaction with PTRF. In this experiment, an N-terminally truncated version of human PTRF, PTRFΔN150, was fused to the B42 transcription activation domain. As a negative control, pRHM1, a plasmid that expresses LexA fused to a transcriptionally inert fragment of the Drosophila melanogaster Bicoid protein (amino acid residues 2–160) was used. Numbers refer to the amino acids within TTF–I and Bicoid which are contained in the respective fusion proteins. Activation of the LacZ reporter gene was quantified by a liquid β-galactosidase assay. The mean values of three independent experiments are shown. (B) PTRF interacts with immobilized TTF-I. Histidine-tagged TTFΔN185 was expressed in baculovirus-infected Sf9 cells (Sander et al., 1996) and immobilized on Ni2+-NTA–agarose beads (Pharmacia). A yeast extract (15 μg of total protein) containing HA-tagged PTRFΔN150 (L) was loaded onto a 10 μl column containing bound TTFΔN185 (lanes 2–5) or control Ni2+-NTA beads (lanes 6–9). The flow-through (FT) fraction, 25% of the yeast extract (L), two wash fractions (W) and the total amount of the 1 M KCl eluate (E) were analyzed on Western blots with anti-HA (12CA5) monoclonal antibodies. (C) Binding of TTF-I to immobilized PTRF. GST or GST–PTRF were expressed in E.coli, bound to glutathione beads, and incubated with 35S-labeled mTTF-I (L). The beads were washed with 10 vols of buffer AM-100 (W) and eluted with high salt buffer AM-1000 (E). Download figure Download PowerPoint In order to demonstrate that TTF-I and PTRF can also interact in vitro, we performed affinity chromatography using either TTF-I or PTRF as immobilized ligands. To monitor binding of PTRF to bead-bound TTF-I, a crude yeast extract containing hemagglutinin (HA)-tagged recombinant PTRF was chromatographed on histidine-tagged TTF-I bound to a nickel-chelate matrix and bound proteins were eluted with salt. The unbound proteins (FT), the wash (W) and the eluate (E) were analyzed on immunoblots using anti-HA antibodies. As shown in Figure 3B, a significant amount of PTRF bound to and could be eluted from the TTF-I beads (lanes 1–5), whereas no binding of PTRF to control beads was observed (lanes 6–9). The reciprocal experiment, i.e. binding of TTF-I to immobilized PTRF, is shown in Figure 3C. A glutathione S-transferase (GST) fusion protein containing the entire ORF of murine PTRF (GST–PTRF) was produced in Escherichia coli, bound to glutathione–Sepharose beads and used to bind TTF-I which was synthesized in rabbit reticulocyte lysates. Approximately 20% of 35S-labeled TTF-I was retained on the GST–PTRF (Figure 3C, lane 5) but not on the GST control resin (Figure 3C, lane 3). Thus, the in vitro binding results confirm the physical interaction between PTRF and TTF-I and extend the observations obtained by the genetic interaction screen in yeast. PTRF associates with RNA polymerase I In previous experiments, we have observed a great deal of variability in the extent of transcript release depending on the Pol I preparation used. We have separated two forms of Pol I chromatographically, one that is competent for transcript release on its own, and one that is release-deficient, but can be complemented by cellular fractions containing PTRF (Mason et al., 1997a). This result suggests that the release factor is associated with and can be dissociated from Pol I. To monitor the interaction between PTRF and Pol I, antibodies against PTRF and the third largest murine Pol I subunit PAF/RPA53 (Seither et al., 1997), respectively, were bound to magnetic beads and incubated with a partially purified protein fraction (DEAE-280) derived from mouse nuclear extracts. Proteins bound to the immobilized antibodies and to control beads, respectively, were analyzed on immunoblots using anti-PTRF and anti-Pol I (α-RPA116) antibodies. As shown in Figure 4, significant amounts of Pol I were co-precipitated with anti-PTRF antibodies (lane 3). In the reciprocal experiment, i.e. co-immunoprecipitation of PTRF with Pol I, we also observed a strong interaction between both proteins (lane 6). Thus PTRF, by interacting with both Pol I and TTF-I, appears to serve a role in mediating the contact between TTF-I and the paused RNA polymerase. Figure 4.Co-immunoprecipitation of PTRF and RNA polymerase I. Partially purified nuclear extract proteins (DEAE-280) were incubated with bead-bound rabbit anti-PTRF antibodies (α-PTRF, lane 3), anti-Pol I antibodies (α-PAF/RPA53, lane 6) and the respective pre-immune sera (Pre, lanes 2, 5). Twenty percent of the DEAE-280 fraction (Load) and the total of precipitated proteins were analyzed on immunoblots with anti-PTRF and anti-RPA116 antibodies. Download figure Download PowerPoint Recombinant PTRF mediates release of both nascent transcripts and RNA polymerase I To prove that the cloned cDNA encodes functionally active PTRF, we tested the recombinant protein in the transcript release assay. For this, PTRF was expressed in E.coli, purified by chromatography on Ni2+-NTA–agarose and S–Sepharose, and assayed in transcription reactions containing immobilized tailed template, Pol I and TTF-I. Clearly, the majority of transcripts were released from the template in the presence of PTRF (Figure 5A, lanes 1–6), whereas no transcript release was observed in reactions containing an unrelated protein (GCN5) which was expressed and purified in parallel. Figure 5.Recombinant PTRF mediates transcript release. (A) Complementation of release-deficient Pol I with recombinant PTRF. Transcript release was assayed in the absence or presence of increasing amounts of recombinant histidine-tagged PTRF or GCN5 as indicated, and the distribution of bound (b) and released (r) transcripts was determined. (B) Recombinant PTRF releases transcripts from washed ternary transcription complexes. Transcription reactions were incubated for 5 min to allow Pol I to reach the terminator. The paused complexes were removed by magnetic attraction, washed with buffer AM-200 to remove free Pol I and nucleotides, and incubated for another 5 min with NTPs and recombinant histidine-tagged PTRF or GCN5 as indicated. Download figure Download PowerPoint A qualitatively similar result was obtained if RNA release was not assayed with release-deficient Pol I, but on isolated paused ternary transcription complexes. Paused complexes were formed by pre-incubating Pol I with the immobilized template, NTPs, Pol I and TTF-I. The remarkable stability of paused transcription complexes allows them to be washed and thus to be depleted of excess Pol I and TTF-I. In the experiment shown in Figure 5B, bead-bound ternary complexes were isolated, washed and then incubated with increasing amounts of recombinant PTRF. Again, in the absence of PTRF, all transcripts remained associated with the template (Figure 5B, lanes 1 and 2) whereas, after addition of PTRF, the transcripts were released into the supernatant (Figure 5B, lanes 3–6). Moreover, consistent with previous results demonstrating that transcript release is an energy-independent process (Mason et al., 1997a), recombinant PTRF promoted dissociation of paused ternary complexes both in the absence of NTPs and in the presence of non-hydrolyzable nucleotides (data not shown). If the recombinant protein exerts the same functional properties as cellular PTRF, then it should facilitate release of not only transcripts but also Pol I from paused elongation complexes. To address this issue, a modified transcription assay containing immobilized Pol I was used. In the experiment shown in Figure 6A, the transcription reactions contained a labeled DNA template, TTF-I, nucleotides and Pol I that was bound to magnetic beads via antibodies against RPA116, the second largest subunit of murine Pol I (Seither and Grummt, 1996). Pol I fixed to magnetic beads is capable of supporting specific transcription (Seither et al., 1998). To monitor dissociation of ternary complexes, transcription was performed with bead-bound Pol I, labeled template, TTF-I and cold nucleotides. Transcription complexes were isolated by magnetic attraction, washed, resuspended in transcription buffer and incubated in the absence and presence of PTRF. Finally, the assays were separated into bead-bound and supernatant fraction, and the distribution of labeled DNA was analyzed. In the absence of PTRF, the template was in the bead-bound fraction, indicating that it was contained within the ternary transcription complex (Figure 6A, lanes 1 and 2). However, in the presence of both recombinant PTRF (Figure 6A, lanes 3–8) and partially purified cellular PTRF (Figure 6A, lanes 9 and 10), the majority of labeled DNA was found in the soluble fraction. This result indicates that PTRF induced dissociation of ternary transcription complexes and therefore liberated the template from bead-bound Pol I. Figure 6.PTRF mediates dissociation of ternary transcription complexes. (A) PTRF-dependent release of template DNA from ternary complexes containing immobilized Pol I. Ternary complexes were formed by pre-incubating bead-bound Pol I with labeled pCAT-T6-T1 template and cold nucleotides. After addition of increasing amounts of recombinant PTRF (lanes 3–8) or cellular PTRF (lanes 9 and 10), the distribution of bound and released template was analyzed. (B) PTRF-dependent release of transcripts from ternary complexes containing immobilized Pol I. Reactions were identical to those described in (A) except that the assays contained unlabeled template and [α-32P]GTP. Download figure Download PowerPoint In parallel reactions, transcript release was measured under the same conditions, except that in these assays the template was not labeled and the reactions were supplemented with [α-32P]GTP. As shown in Figure 6B, in the presence of PTRF, liberation of transcripts into the supernatant was observed, demonstrating that PTRF promotes transcript release irrespective of whether the template or the polymerase were fixed to magnetic beads. This tight correlation between PTRF-dependent release of both template DNA and nascent transcripts from immobilized ternary Pol I complexes demonstrates that PTRF is capable of dissociating stalled ternary complexes, thereby liberating both Pol I and RNA. PTRF binds specifically to the 3′ end of pre-rRNA As shown above, cellular PTRF binds to the 3′ end of pre-rRNA, and this binding appears to be required for transcript release. To establish whether recombinant PTRF has the same specificity with respect to binding to the U-rich element, we first compared the wild-type (pCAT-T6-T1) and the mutant template (pCAT-G6-T1) in transcript release assays using histidine-tagged PTRF expressed in E.coli. Consistent with the requirement for the U stretch in PTRF function, recombinant PTRF mediates transcript release from the wild-type (Figure 7A, lanes 1–6) but not from the mutant template (Figure 7A, lanes 7–12). Furthermore, like the cellular factor (Figure 1), recombinant PTRF binds to an RNA probe harboring 3′-terminal pre-rRNA sequences (Figure 7B, lanes 1–3), and substitution of the U stretch by G residues strongly impairs binding (Figure 7B, lanes 4–6). This result underscores the importance of the U run at the 3′ end of pre-rRNA for dissociation of TTF-I-stalled ternary transcription complexes and demonstrates that the recombinant protein exhibits the same functional properties as the cellular factor. Figure 7.Recombinant PTRF requires the T stretch upstream of the T1 terminator. (A) Recombinant PTRF releases transcripts from wild-type but not mutant templates. Transcriptions were performed on bead-bound pCAT-T6-T1 (lanes 1–6) and pCAT-G6-T1 (lanes 7–12) in the absence or presence of recombinant histidine-tagged PTRF as indicated, and fractionated into template-bound (b) and released (r) transcripts. (B) Recombinant PTRF recognizes the nucleotide sequence of the 3′ end of pre-rRNA. Histidine-tagged PTRF was incubated with labeled RNA representing either the wild-type (lanes 1–3) or mutant version (lanes 4–6) of the 3′ end of pre-rRNA. The reactions were resolved on a 5% polyacrylamide gel and visualized by autoradiography. Download figure Download PowerPoint Discussion Transcription termination by Pol I is a multistep process involving pausing of the elongating polymerase, release of both the newly synthesized RNA and Pol I, and 3′-end processing of the primary transcript (Reeder and Lang, 1994, 1997; Mason et al., 1998). Despite great differences in the sequences of the terminator elements and the DNA-binding proteins from species as diverse as mouse, frog and yeast, the mechanism of termination in all eukaryotes is probably very similar. All characterized Pol I terminator elements are recognized by a specific DNA-binding protein that either directly or indirectly contacts the elongating RNA polymerase and mediates the termination reaction. With the availability of cloned terminator proteins, it has been possible to establish cell-free transcription systems which terminate Pol I at the same sites as utilized in vivo and thus allow the study of the mechanism of transcription termination. These studies revealed that Pol I transcription termination can be separated into two mechanistically distinguishable steps. First, Pol I is paused by a DNA-bound protein, e.g. in the mouse by the transcription termination factor TTF-I bound to the 'Sal box' te