INTRODUCTION

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Transcription elongation has been found to be an important target for control of eukaryotic gene expression (1, 2). After initiating from a promoter, the RNA polymerase II elongation complex is subjected to a block to elongation by the action of components of N-TEF (negative transcription elongation factors).¹ One component of N-TEF, Drosophila factor 2, was originally identified by its ability to suppress the generation of long RNA polymerase II transcripts (3). Later it was found that factor 2 caused termination of transcription in an ATP-dependent manner (4-6). DSIF was identified as another negative factor comprised of the human homologues of yeast SPT4 and SPT5 (7, 8). The action of P-TEFb counters the negative factors and allows long transcripts to be made (9), presumably through its ability to phosphorylate the carboxyl-terminal domain of the large subunit of RNA polymerase II (10). After the polymerases have made the transition into a productive mode, elongation is made more efficient by the action of other general elongation factors such as TFIIF and S-II (2, 11).

It has been reported that RNA polymerases can undergo spontaneous termination at specific sites along the template; however, efficient and accurate termination at other sites requires accessory protein factors (11, 12). These proteins function through several distinct mechanisms. One involves an RNA helicase that binds to and tracks along nascent RNA chains. When it encounters a transcription complex at a termination site, it disrupts the RNA-DNA hybrid causing release of the RNA. Rho protein for Escherichia coli RNA polymerase and La protein for eukaryotic RNA polymerase III belong to this category (13-15). Another termination mechanism involves two components. One component binds to a specific DNA sequence and serves as a "roadblock" to elongation by RNA polymerase. The other then terminates the stalled complexes in an energy-independent manner. TTF-I, Reb1p, and a termination activity for RNA polymerase I belong to this category (16-18). The third mechanism involves protein components that bind to DNA and/or RNA polymerase and employ DNA-dependent ATPase activity to dissociate polymerase complexes from the DNA template. VTF/CE and NPH-I proteins for vaccinia polymerase function through this mechanism (19, 20). Previous work in our laboratory indicates that factor 2 causes termination of RNA polymerase II in a similar fashion (4, 5).

	EXPERIMENTAL PROCEDURES

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Cloning, Expression, and Production of Antibodies to Drosophila Factor 2-- Approximately 85 pmol (13 µg) of purified Drosophila factor 2 (DmF2) (4) were excised after SDS-PAGE and subjected to peptide sequencing. The three peptide sequences obtained (NLSQPTIQAVLK, YRWALTGTPIQNK, and KLDLADGVLTGAK) were identical to regions in a known protein, lodestar. A cDNA encoding lodestar was amplified by PCR from a Drosophila embryonic cDNA mix (CLONTECH) and cloned into a pET21a vector (Novagen). The plasmid was expressed in DE3 cells grown to an A₆₀₀ of 0.6 and induced with 0.5 mM isopropyl--D-thiogalactopyranoside for 1.5 h. Cells were then harvested and resuspended in 100 mM HGKEDP (25 mM HEPES (pH 7.6), 15% glycerol, 100 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, and 0.1% of a saturated solution of PMSF in isopropanol). Cells were lysed by passing through a French press three times at 4 °C. The cell lysate containing rDmF2 was subjected to centrifugation at 15,000 × g for 20 min, and the supernatant was loaded onto a 30-ml P-11 column equilibrated with 0.1 M HGKEDP. Proteins were eluted with 0.3 and 1 M HGKEDP. The 1 M P-11 step was dialyzed against 0.1 M TUS (10 mM Tris (pH 7.5), 7 M urea, and 0.1 M NaCl) and applied to a 1-ml Mono Q column equilibrated with 0.1 M TUS. Proteins bound to Mono Q were eluted with 0.1-1 M NaCl gradient in TUS buffer. Fractions containing rDmF2 (0.18-0.3 M NaCl) were pooled, dialyzed, and loaded onto a 1-ml Mono S column equilibrated with 0.1 M NaCl in TUS buffer. The column was then eluted with 0.1-0.6 M NaCl gradient in TUS buffer. Fractions containing factor 2 (0.12-0.18 M NaCl) were pooled, dialyzed against phosphate-buffered saline, and used to immunize rabbits (Pocono Rabbit Farm & Laboratory, Inc.).

Cloning of Human Factor 2-- We searched the EST data base for possible homologues using BLAST programs (21) and identified several human ESTs. I.M.A.G.E. Consortium Clone 357834 (22) contained the 3' end of human factor 2. Using the sequence of this clone and another EST (70720) primers were designed to obtain the 5' end of the coding region from Marathon-Ready human brain cDNA (CLONTECH). Single strand cDNA was synthesized from HeLa total RNA using a primer downstream of the stop codon. A double-stranded cDNA encoding human factor 2 (HuF2) was amplified by PCR and cloned into the modified expression vector pBAC4X-1 (Novagen) to create plasmid pBhHF that encodes an NH₂-terminally (His)₆-tagged human factor 2.

Sequence Analysis-- Pairwise or multiple alignments of full-length factor 2 proteins or their conserved COOH-terminal domains (amino acids 415-1061 for DmF2, 365-1026 for Caenorhabditis elegans factor 2, and 374-990 for HuF2) were made by Gap, Bestfit, and Pileup programs of the GCG program package. The primary sequence analysis was done using SAPS program (23). Secondary structure predication was done with PhD program (24). The nuclear localization sequence was predicted by PSORT program combined with sequence alignment. PEST sequence search was done using PESTfind program (25).

Expression and Purification of Human Factor 2-- Recombinant baculoviruses were generated using BaculoGold DNA (PharMingen) and pBhHF plasmid. Sf9 cells were harvested 3 days postinfection and were lysed for 40 min in lysis buffer H (10 mM Tris-HCl at pH 8.0, 150 mM NaCl, 2 mM MgCl₂, 20 mM -mercaptoethanol, 1% Triton X-100, and 0.1% of a saturated solution of PMSF in isopropanol). After centrifugation at 2,000 g for 15 min, the lysate was brought to 1 M NaCl and 5 mM imidazole before a 45 min centrifugation at 250,000 × g. The cleared lysate was loaded onto a Ni²⁺-nitrilotriacetic acid-agarose column (Qiagen). The column was washed with a solution containing 1 M NaCl, 10 mM Tris-HCl (pH 8.0), 5 mM imidazole, 10% glycerol, and PMSF and then washed with a similar buffer containing 0.1 M NaCl. HuF2 was eluted by raising the imidazole to 200 mM. After diluting the fractions to 65 mM NaCl using HGEDP (25 mM HEPES at pH 7.6, 15% glycerol, 0.1 mM EDTA, 1 mM dithiothreitol, 0.1% of a saturated solution of PMSF in isopropanol) the sample was cleared by 30-min centrifugation at 2,000 × g and loaded onto a 1-ml Mono S column. A linear gradient from 0.065-0.5 M HGKEDP was used to elute the recombinant protein. The peak fractions (0.11-0.24 M) were pooled, diluted to 0.12 M HGKEDP, and cleared as described above before being loaded onto a 1-ml Mono Q column. The recombinant protein eluted from 0.20 to 0.27 M by a linear gradient from 0.12 to 0.4 M HGKEDP.

ATPase Assay and Transcription Termination Assay-- ATPase assays and transcription termination assays using Drosophila K_c cell nuclear extract and actin 5C template were carried out essentially as described previously (4, 5). The immobilized CMV template was generated by first cloning the KpnI/HindIII fragment containing the CMV promoter into pGL2-basic (Promega) to generate pGL2-CMV. After digesting this plasmid with BamHI the resulting DNA fragments were end-filled using Klenow, dCTP, dGTP, dTTP, and biotin-14-dATP. The DNA was digested with EcoRI to produce a 631-nucleotide runoff transcript, phenol-extracted, ethanol-precipitated twice, and dissolved in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) (pH 8.0). The DNA solution was brought to 0.5 M NaCl and incubated with Streptavidin-coated beads (Dynal) for 30 min (~40 µg/mg beads). For the termination assay the immobilized CMV template (final concentration ~50 µg of DNA/ml) was incubated with HeLa nuclear extract in the presence of 20 mM HEPES (pH 7.6) and 7 mM MgCl₂ for 10 min at 30 °C. The preinitiation complexes were pulse-labeled for 25 s. The early elongation complexes were washed three times with 1 M HMK (20 mM HEPES, 5 mM MgCl₂, and 1 M KCl) + 1% Sarkosyl (~4 min for each wash). The complexes were then washed four times with 65 mM HMKB (20 mM HEPES, 7 mM MgCl₂, 65 mM KCl, and 0.2 mg/ml bovine serum albumin). The beads were resuspended in 65 mM HMKB solution and assayed for termination as described (4). The RNA transcripts were assayed on 15% denaturing polyacrylamide gel and quantitated using Packard InstantImager.

	RESULTS

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Cloning of Drosophila and Human Factor 2-- To further our understanding of the molecular basis of the termination activity of factor 2, we wanted to identify the gene encoding the factor. Sequence information was obtained from purified Drosophila factor 2 (DmF2), and a data base search indicated that it was a known protein, lodestar. Lodestar was first identified as a maternal-effect protein involved in chromosome segregation during mitosis (26). Probing data bases with the Drosophila protein sequence yielded several human ESTs and a cDNA encoding an uncharacterized C. elegans protein. A full-length cDNA encoding the putative human homologue (HuF2) was generated, and its sequence was compared with Drosophila factor 2 and the putative C. elegans homologue² (Fig. 1). The three proteins shared seven motifs found in helicase superfamilies 1 and 2 and seven additional motifs shared only by SWI2/SNF2 family members (Fig. 1). The full-length DmF2 and HuF2 have 43% identity and 62% similarity, while their more highly conserved carboxyl-terminal regions are 48% identical and 68% similar, significantly higher than the average 31% identity and 53% similarity shared among SWI2/SNF2 family members (27). The putative C. elegans homologue shares similar identity to both HuF2 and DmF2. The three proteins also share three conserved motifs and a predicated nuclear localization signal (between helicase motifs I and Ia) that are not conserved among the SWI2/SNF2 family (Fig. 1). Unlike DmF2 (26), HuF2 has no predicted PEST sequence. Except for the COOH-terminal domain and the three factor 2 motifs there is little additional sequence conservation in these three proteins. The NH₂ termini of the factor 2 proteins are predicted to be relatively unstructured compared with the highly structured "helicase" domains. The predicted secondary structures of the factor 2-specific motifs are also conserved. Motif B shows high sequence similarity (56% identity and 68% similarity) to a 24-amino acid fragment of a poliovirus capsid protein and is predicted to have secondary structure throughout the region. However, the functional significance of this potential structural homology is not known.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Factor 2 belongs to SWI2/SNF2 DNA-dependent ATPase family. The sequences of Drosophila, human, and a putative C. elegans factor 2 were aligned. Specific protein motifs are shown as indicated in the legend. The percent identity between the factor 2 homologues is indicated.

Human Factor 2 Has dsDNA-dependent ATPase Activity-- To confirm that the cloned cDNAs encoded factor 2, we generated recombinant forms of both the Drosophila and the human proteins. DmF2 was expressed in E. coli, but the resulting proteins were mostly truncated forms and were insoluble. The longest forms of the recombinant DmF2 had an identical mobility on SDS-PAGE to factor 2 purified from K_c cells (data not shown). Antibodies raised against the recombinant Drosophila protein reacted strongly and specifically with authentic Drosophila factor 2 either in purified form (Fig. 2A) or in crude K_c cell nuclear extract (data not shown). This provides strong evidence that the lodestar protein is DmF2. HuF2 was expressed in a baculovirus expression system as a soluble, NH₂-terminally (His)₆-tagged protein. The purified recombinant HuF2 was compared with factor 2 purified from K_c cells by silver-stained SDS-PAGE (Fig. 2B). The concentration of DmF2 was known, and the concentration of HuF2 was estimated by densitometric comparison of the two proteins on the stained gel.

View larger version (79K): [in this window] [in a new window]   Fig. 2.   Analysis of Drosophila and recombinant human factor 2. A, increasing amounts (2.1-8.4 ng) of purified DmF2 was detected in Western blot using antibodies raised against recombinant Drosophila lodestar protein. The primary and secondary antibodies were used at 1:20,000 dilution. B, increasing amounts of DmF2 (21-84 ng) and baculovirus-expressed recombinant HuF2 (2-8 µl) were analyzed on a silver-stained 6-15% gradient SDS-PAGE gel.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   The dsDNA-dependent ATPase activities of DmF2 and HuF2 are suppressed by ssDNA. A, the ATPase activity of DmF2 (A) and HuF2 (B) were titrated in the presence of 20 ng/µl linearized dsDNA (A5C template restricted with HpaI). C, the ATPase activities of DmF2 and HuF2 were determined in the presence of increasing amounts of dsDNA. D, the dsDNA-dependent ATPase activities of DmF2 and HuF2 were determined in the presence of 20 ng/µl dsDNA and increasing amounts of ssDNA (poly(dT)1,000). In C and D more HuF2 (17.3 ng) was used than DmF2 (4.2 ng) to balance the ATPase activities.

Human Factor 2 Has Transcription Termination Activity-- We further tested HuF2 in transcription termination reactions. The assay detects the release of transcripts from early elongation complexes formed on an immobilized template into the supernatant fraction. Increasing amounts of both DmF2 and HuF2 were incubated with early elongation complexes formed using either Drosophila (Fig. 4A) or human (Fig. 4B) transcription factors. Both factors were able to cause release of the transcripts in an ATP-dependent manner, and the termination activity correlated with the amount of protein added. Surprisingly, even though HuF2 had a slightly weaker ATPase activity compared with DmF2, it caused the release of nascent Drosophila RNA polymerase II transcripts as efficiently (Fig. 4A). HuF2 released nascent human RNA polymerase II transcripts roughly 3-fold more efficiently than DmF2 (Fig. 4B). The activity of both factors exhibited no preference for transcript length. When added to saturating level, these two proteins were able to release over 80% RNA transcripts in both systems.

View larger version (63K): [in this window] [in a new window]   Fig. 4.   DmF2 and HuF2 possess ATP-dependent termination activity and function on human and Drosophila RNA polymerase II elongation complexes. The termination assay diagrammed at the top of the figure was followed for Drosophila (A) and human (B) RNA polymerase II early elongation complexes. Released transcripts (supernatant) were analyzed on a 15% denaturing polyacrylamide gel. The amounts of DmF2 and HuF2 added into the reaction were increased at an interval of 3-fold to the highest amount of 21 and 17.3 ng respectively. ATP was included in the reaction where indicated. "Total" represents transcripts bound to beads in the starting material.

	DISCUSSION

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We have extended our studies of factor 2 by cloning the Drosophila and human factor and generating functional recombinant human factor 2. We found factor 2 belongs to the SWI2/SNF2 family of proteins that are involved in modulating protein-DNA interactions during transcription, recombination, and repair. Unique biochemical properties and protein sequence motifs distinguish factor 2 from other SWI2/SNF2 family members and all other termination factors.

Of all the known termination factors factor 2 is most similar to NPH-I, a viral protein required for VTF/CE-mediated termination of vaccinia polymerase (19, 20). Both factor 2 and NPH-I are ATPases, but NPH-I is stimulated by ssDNA and factor 2 by dsDNA. In the absence of a termination sequence detected by VTF/CE, NPH-I stimulates transcription elongation of vaccinia RNA polymerase (19), but factor 2 has no positive effect on elongation. Comparison of the sequences of the two proteins indicates that they are similar in that they both contain the seven helicase motifs while not exhibiting helicase activity. However, NPH-I contains no SWI2/SNF2 or factor 2-specific motifs and cannot function independently of VTF/CE. Although there are significant differences between the two proteins, further biochemical characterization is required to determine whether they share any steps in their mechanisms of termination.

Comparison of the sequence of factor 2 to the other approximately 150 SWI2/SNF2 family members allows several hypotheses to be made concerning the function of factor 2 and SWI2/SNF2 proteins in general. The ATPase activity of factor 2 is likely encoded by its carboxyl terminus that contains the seven helicase motifs and SWI2/SNF2 motifs shown to be responsible for the ATPase activity and required for the function of other family members (28, 29). The three factor 2-specific motifs surrounding the ATPase domain may be involved in specifying the unique nucleic acid binding properties of the factor or may be involved in specifically coupling ATP hydrolysis to polymerase termination. This idea is supported by the finding that a region outside of the ATPase domain of Mot1p confers its functional specificity (28). Analysis of factor 2 proteins with mutations in these regions using DNA binding and termination assays may be able to resolve these possibilities. It has been suggested that a common feature of SWI2/SNF2 proteins is that they use ATP hydrolysis to disrupt protein-DNA interaction (30), e.g. SNF2 disrupts nucleosome-DNA interactions and MOT1 removes TBP from DNA. The identification of factor 2 as a SWI2/SNF2 family member provides further evidence supporting the ATP driven disruption hypothesis, since the protein couples ATP hydrolysis with removal of RNA polymerase II from the DNA template.

Based on the biochemical characterization of Drosophila factor 2 we suggested that it might be the eukaryotic equivalent of the prokaryotic transcription repair coupling factor (TRCF), Mfd (4-6). Mfd causes termination of RNA polymerase stalled over damaged DNA and then recruits the ABC repair machinery to the damage site (31, 32). The cloning of factor 2 extends the similarity between the two proteins, since both contain helicase motifs. ERCC6 was suggested to be the human TRCF (33); however, several lines of evidence argue against this notion. Mutants of the yeast and mouse homologues of ERCC6 do not affect UV sensitivity (27, 34). ERCC6 cannot dissociate stalled RNA polymerase from the DNA template (35). More decisively, ERCC6 was dispensable for transcription-coupled repair in vitro (36). Although the role of factor 2 as the TRCF is consistent with previous biochemical characterization and the sequence data presented here, it will be necessary to examine the role of factor 2 in defined transcription-coupled repair assays.

The identification of Drosophila factor 2 as the maternal-effect protein, lodestar, may help to explain several previous observations. Lodestar mutants die during early embryogenesis and display chromosome segregation defects. As suggested previously, a reduction in DNA repair could result in this phenotype (26). If so, this supports a role of factor 2 in transcription-coupled repair. Alternatively, chromosome segregation may be hindered by the presence of active transcription complexes during mitosis. Mitotic disruption of elongation complexes on long genes such as Ubx has been observed (37). Consistent with a role of factor 2 in this process, its localization in the nucleus (26) coincides with the mitotic termination of Ubx transcription (37). The possible involvement of factor 2 in elongation control, transcription-coupled repair, and mitotic termination suggests that the activity of factor 2 must be carefully regulated.