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J Biol Chem, Vol. 275, Issue 17, 12393-12399, April 28, 2000


Transcription Factor S, a Cleavage Induction Factor of the Archaeal RNA Polymerase*

Winfried HausnerDagger , Udo Lange, and Meike Musfeldt

From the Institut für Allgemeine Mikrobiologie, University of Kiel, Am Botanischen Garten 1-9, D-24118 Kiel, Federal Republic of Germany

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have analyzed the function of an archaeal protein (now called transcription factor S (TFS)) that shows sequence similarity to eukaryotic transcription factor IIS (TFIIS) as well as to small subunits of eukaryotic RNA polymerases I (A12.6), II (B12.2), and III (C11). Western blot analysis with antibodies against recombinant TFS demonstrated that this protein is not a subunit of the RNA polymerase. In vitro transcription experiments with paused elongation complexes at position +25 showed that TFS is able to induce cleavage activity in the archaeal RNA polymerase in a similar manner to TFIIS. In the presence of TFS, the cleavage activity of the RNA polymerase truncates the RNA back to position +15 by releasing mainly dinucleotides from the 3'-end of the nascent RNA. Furthermore, TFS reduces the amount of non-chaseable elongation complexes at position +25 as well as position +45. These findings clearly demonstrate that this protein has a similar function to eukaryotic TFIIS.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Considerable progress has been made recently in the functional analysis of initiation of transcription in Archaea. It seems to be clear now that initiation of transcription in Archaea is mediated by two basal transcription factors, archaeal TATA box-binding protein (aTBP)1 and TFB, which are orthologous to the eukaryotic TATA box-binding protein and TFIIB (reviewed in Refs. 1-3). These two factors together with RNA polymerase are necessary and sufficient for initiation of in vitro transcription at some promoters (4, 5). These findings are in line with the data of genome sequencing projects in Archaea. Analysis of the genomes of Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Pyrococcus horikoshii, and Archaeoglobus fulgidus indicated that there are, with the exception of a putative alpha -subunit of TFIIE, no further eukaryote-like initiation factors corresponding to TFIIA, TFIIF, or TFIIH (6-9). Interestingly the genome sequences revealed the presence of a putative transcription factor, which could be involved in the elongation of transcription.

The sequence of this protein was first identified in Sulfolobus acidocaldarius and was suggested to be a homologue to the eukaryotic elongation factor TFIIS (10). Identification of an additional candidate in Thermococcus celer and a detailed analysis of the sequence similarity of these proteins to eukaryotic counterparts revealed that the archaeal TFIIS-like protein is much more likely to be a subunit of the archaeal RNA polymerase than a transcription factor with a similar function to eukaryotic TFIIS (3, 11-14). Several lines of evidence support this suggestion. First, the archaeal genes code for proteins that are ~120 amino acids in size, whereas the eukaryotic TFIIS proteins are much larger (~300 amino acids). Second, the archaeal proteins contain two putative zinc-binding domains instead of the single domain found in eukaryotic TFIIS proteins. Third, eukaryotic RNA pol I, II, and III contain related subunits (A12.6, B12.2, and C11) of similar size and two zinc-binding motifs. Fourth, owing to the close evolutionary relationship of the archaeal and eukaryotic RNA polymerases, one would expect to find a homologous subunit in the archaeal RNA polymerase.

Since there are no biochemical data available at this time about the role of these proteins in Archaea, we have analyzed the function of the archaeal TFIIS/RPSU (RNA polymerase subunit) homologue in a Methanococcus thermolithotrophicus cell-free transcription system. We show here that this protein is not associated with the RNA polymerase, but is able to induce RNA cleavage in the archaeal RNA polymerase similar to eukaryotic TFIIS.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents and Enzymes-- [gamma -32P]ATP and [alpha -32P]UTP were purchased from Hartmann Bioanalytics (Braunschweig, Germany). Restriction endonucleases and other DNA-modifying enzymes were purchased from Fermentas or New England Biolabs Inc.

Templates for in Vitro Transcription-- The plasmid pIC-31/30PRO-C25 used in this study is based on the tRNAVal promoter of Methanococcus vannielii (15). The cytosine residues in the region from positions +2 to +25 were substituted by other bases (see Fig. 3A) using PCR and the plasmid pIC-31/30 as template. The forward primer was complementary to sequences ~120 bp upstream of the transcription start site, and the reverse primer was partly complementary to sequences from positions -4 to +41. After hydrolyzing the amplified fragment with HindIII, a 90-bp fragment containing the promoter and the mutated region downstream of the transcription start site of the tRNAVal gene was isolated and cloned between the HindIII and SmaI (compatible to the blunt ends on one site of the fragment produced by PCR amplification) restriction sites of the vector pIC-20H. The resulting plasmid, pIC-31/30PRO-C25, allowing transcription in the absence of CTP until position +25, was used to generate an ~500-bp transcription template by PCR amplification. Oligonucleotides complementary to sequences ~380 bp upstream of the start site and ~120 bp downstream were used as primers. The upstream primer was labeled with biotin, and the resulting fragment was attached to streptavidin magnetic beads (Roche Molecular Biochemicals) according to the manufacturer's protocol. The immobilized DNA templates were used in all transcription experiments except that presented in Fig. 1.

Plasmid pIC-31/30PRO-C45, allowing transcription in the absence of CTP until position +45, was constructed using PCR and plasmid pIC-31/30PRO-C25 as template. The cytosine residues in the region from positions +26 to +45 were mutated by using the primer 5'-TGAATTCGAGCTCGGTAACCCCATTATTCAAATTTACTTACATT-3' and a reverse primer complementary to sequences ~380 bp upstream of the start site. The amplified insert was hydrolyzed with SapI and EcoRI and inserted between the SapI and EcoRI restriction sites of plasmid pIC-31/30PRO-C25 to generate pIC-31/30PRO-C45.

Purification of the RNA Polymerase-- RNA polymerase from Mc. thermolithotrophicus was purified as described previously (16).

Expression and Purification of Recombinant aTBP-- The coding region of aTBP (GenBankTM/EBI accession number AJ271331) was subcloned using PCR amplification to generate the coding region with an NdeI restriction site at the 5'-end of the sequence and a BamHI site at the 3'-end. The amplified insert was then cloned between the NdeI and BamHI restriction sites of the pET14b expression vector to generate pTBPMth.14, allowing expression of aTBP with an N-terminal hexahistidine tag. BL21(DE3) pLysS cells containing the pTBPMth.14 plasmid were grown to A600 = 0.8 at 25 °C. Expression of the proteins was induced by addition of 1 mM isopropyl-1-thio-beta -D-galactopyranoside. Cells were harvested by centrifugation 3 h after induction, resuspended in buffer (50 mM Tris, pH 8.0, 50 mM NaCl, and 20% glycerol), and disrupted by passage through a French press cell. The lysate was clarified by centrifugation at 4 °C (100,000 × g for 20 min), and aTBP was purified by Ni2+-nitrilotriacetic acid-agarose (QIAGEN Inc.), MonoQ (Amersham Pharmacia Biotech), and Superdex 200 (Amersham Pharmacia Biotech) chromatography. The purified proteins were analyzed by SDS-polyacrylamide gel electrophoresis and stored at -70 °C.

Expression and Purification of Recombinant TFB-- The coding region of TFB (GenBankTM/EBI accession number AJ271467) was subcloned using PCR amplification to generate the coding region with an NdeI restriction site at the 5'-end of the sequence. The amplified insert was then cloned between the NdeI and EcoRV restriction sites of the pET17b expression vector to generate pTFBMth.17. BL21(DE3) cells containing the pTFBMth.17 plasmid were grown to A600 = 0.8 at 37 °C. Protein expression and preparation of crude extract with buffer (50 mM Tris, pH 7.5, and 300 mM NaCl) were done as described for aTBP. TFB was purified by HiTrap heparin (Amersham Pharmacia Biotech) and Superdex 200 chromatography.

Expression and Purification of the Recombinant TFIIS/RPSU Homologue-- Construction of the expression clones pTFSMth.14 and pTFSMth.17 (GenBankTM/EBI accession number AJ271332) was done as described above, the first one allowing expression of TFIIS/RPSU with an N-terminal hexahistidine tag and the second one without N-terminal fusion. Protein expression and preparation of crude extract with buffer (20 mM Tris, pH 8.0, 50 mM NaCl, and 1 mM dithiothreitol) were also done as described before. After heat treatment (85 °C, 15 min) followed by centrifugation, the recombinant TFIIS/RPSU homologue remained in the supernatant and was further purified by MonoQ (pTFSMth.17) or Ni2+-nitrilotriacetic acid-agarose (pTFSMth.14) and Superdex 200 (both) chromatography.

Preparation of Antibodies against the Recombinant TFIIS/RPSU Homologue-- The Purified untagged TFIIS/RPSU homologue (500 µg) was used for immunization of a rabbit. Immunization was performed by Eurogentec (Seraing, Belgium) following a standard immunization protocol. The anti-TFIIS/RPSU IgG fractions were isolated from the serum by affinity chromatography on protein A-Sepharose (Amersham Pharmacia Biotech). The anti-TFIIS/RPSU IgG fraction was further purified by affinity chromatography on a column with the covalently fixed recombinant TFIIS/RPSU homologue.

Western Blot Analysis-- Western blot analysis was performed as described previously (17).

In Vitro Transcription Reactions-- In vitro transcription experiments were formed in 25-µl reaction mixtures that contained 190 fmol of template, 0.8 pmol of purified RNA polymerase, 1.7 pmol of recombinant aTBP, 1.7 pmol of recombinant TFB, 40 µM ATP/GTP/CTP, and 4 µM [alpha -32P]UTP (370 Bq/pmol) in transcription buffer (20 mM Tris, pH 8.5 at 20 °C, 2 mM MgCl2, 0.1 mM EDTA, 40 mM KCl, and 3 mM dithiothreitol) for 30 min at 55 °C. The reaction was terminated by adding 12.5 µl of loading buffer (98% formamide, 10 mM EDTA, 0.1% bromphenol blue, and 0.1% xylene cyanol). Analysis of transcripts and nonspecific transcription were performed as described previously (16).

Immobilized Transcription-- Ternary complexes stalled at position +25 (see Fig. 3) were formed in 250-µl reaction mixtures that contained 1 pmol of template pIC-31/30PRO-C25 bound to magnetic beads, 8 pmol of purified RNA polymerase, 17 pmol of recombinant aTBP, and 17 pmol of recombinant TFB in transcription buffer. After a 60-min preincubation at 21 °C, the reaction was started by addition of 40 µM ATP/GTP and 4 µM [alpha -32P]UTP (370 Bq/pmol). After 20 min at 55 °C, the supernatant was separated from the magnetic beads; and the beads were washed twice with WAC buffer (20 mM Tris, pH 8.5, 0.1 mM EDTA, 40 mM KCl, 3 mM dithiothreitol, and 0.1% N-lauroylsarcosine), resuspended in 200 µl of 1.25-fold transcription buffer, distributed in 20-µl fractions, and supplemented with the components indicated on top of each figure in a total volume of 25 µl to achieve 1-fold transcription buffer. After further incubation for cleavage, the reactions were terminated as described before. In some cases, aliquots of the reactions were chased before termination by addition of 40 µM unlabeled nucleotides and further incubation for 10 min at 55 °C.

Markers-- RNA size markers were formed by in vitro transcription in the presence of 3'-dATP (100 µM). UpG (250 µM) was labeled at 37 °C for 30 min in a volume of 20 µl with T4 polynucleotide kinase (0.5 unit/µl) and [gamma -32P]ATP (9000 Bq/µl) in 50 mM Tris, pH 7.6, 10 mM MgCl2, and 5 mM dithiothreitol. The reactions were diluted in transcription and loading buffers.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The TFIIS/RPSU Homologue Is Not Necessary for in Vitro Transcription-- To investigate the function of the archaeal TFIIS/RPSU homologue, we first cloned and sequenced the corresponding gene from Mc. thermolithotrophicus. This gene was then expressed in Escherichia coli. The purified recombinant proteins with (lane 1) and without (lane 2) an N-terminal histidine tag are shown in Fig. 1A. To address the question of whether this protein is a component of the Methanococcus in vitro transcription system (18), all components of the cell-free system were challenged with antibodies raised against the recombinant TFIIS/RPSU homologue. Western blot analysis showed that the antibody against the recombinant TFIIS/RPSU homologue bound to a polypeptide in the crude extract of the same size as the recombinant protein (Fig. 1B, lanes 4 and 5), demonstrating that this protein is expressed in Methanococcus. No binding of the antibodies was observed in the samples containing the purified RNA polymerase or, as expected, recombinant transcription factor aTBP or TFB (lanes 1-3). Since these three components are sufficient for in vitro transcription of the tRNAVal gene (Fig. 1C, lanes 1-4), this protein is not an essential component for standard in vitro transcription. Addition of this protein to in vitro transcription reactions slightly decreased RNA synthesis (compare lanes 5-8 with 1-4).


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  		Fig. 1.   The TFIIS/RPSU homologue is not necessary for in vitro transcription. A, purified TFIIS/RPSU homologues with (lane 1) and without (lane 2) an N-terminal histidine tag were analyzed on a 15% SDS/Tricine-polyacrylamide gel. B, 100 ng of recombinant aTBP, 100 ng of recombinant TFB, 1 µg of RNA polymerase, 5 µg of crude extract, and 40 ng of recombinant TFIIS/RPSU homologue were electrophoresed on a 15% SDS/Tricine-polyacrylamide gel; transferred to nitrocellulose membrane; and challenged with a purified IgG fraction raised against the recombinant TFIIS/RPSU homologue. Binding of antibodies was detected with peroxidase-coupled antibodies. C, transcription reactions contained aTBP, TFB, RNA polymerase, and template pIC-31/2. The presence (+) or absence (-) of 10 pmol of TFIIS/RPSU is indicated on top of the lanes. The individual incubation (Inc.) times are also indicated on top of the lanes.

The TFIIS/RPSU Homologue Is Not a Subunit of the RNA Polymerase-- The RNA polymerase used for in vitro transcription is purified by four chromatographic steps (16). Therefore, it is possible that the archaeal TFIIS/RPSU homologue was lost during the purification procedure. To exclude this possibility, we chromatographed crude cell-free extract on a gel filtration column and analyzed the different fractions for RNA polymerase activity and for the presence of the archaeal TFIIS/RPSU homologue. The bulk of the RNA polymerase with a molecular mass of >400 kDa was eluted in fractions 23-27, as analyzed by a nonspecific transcription assay (Fig. 2A). Western blot analysis of fractions 21-39 using an antibody against the largest subunit of the RNA polymerase confirmed the profile of the nonspecific RNA polymerase assay (Fig. 2A). In contrast, the bulk of the TFIIS/RPSU homologue eluted in fractions 33-37, as shown by Western blot analysis (Fig. 2B, compare lanes 7-9 with the lane containing the recombinant TFIIS/RPSU homologue). An additional protein that was also recognized by the anti-TFIIS/RPSU antibody in the crude extract is labeled with a question mark. Since the RNA polymerase and the homologue eluted in different fractions, we assume that this protein is not a subunit of the RNA polymerase.


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  		Fig. 2.   The TFIIS/RPSU homologue is not associated with the RNA polymerase. Fractions eluted by gel filtration chromatography (Superdex 200) were assayed for RNA polymerase activity and for the presence of the TFIIS/RPSU homologue. RNA polymerase activity was analyzed by a nonspecific transcription assay as well as by Western blotting (16). A, the results of the nonspecific transcription assay are indicated on top of the lanes, and the results of the Western blotting using an RNA polymerase antibody raised against the largest subunit (M. Thomm, personal communication) are shown. B, the presence of the TFIIS/RPSU homologue was analyzed by Western blotting as described in the legend to Fig. 1. For the preparation of the fractions, 40 mg of crude extract were loaded onto a Superdex 200 column and collected as 2-ml fractions. 10 µl of these fractions were used for RNA polymerase activity assay, and 20 µl were used for Western blot analysis. The control lanes with purified RNA polymerase, crude extract, and the recombinant TFIIS/RPSU homologue contained 200 ng, 25 µg, and 40 ng of protein, respectively. Preparation of the crude extract and conditions for gel filtration chromatography were as described previously (16). The RNA polymerase and the TFIIS/RPSU-induced signals are marked by arrows. Inc., incubation.

Formation of Stalled Elongation Complexes-- The function of eukaryotic TFIIS has been analyzed by incubating stalled elongation complexes with TFIIS in the absence of nucleotides. For similar analysis of the archaeal TFIIS/RPSU homologue, we have set up a system allowing stalling of the elongation complex on the tRNAVal promoter at position +25. The template was immobilized on magnetic beads using a streptavidin-biotin linkage; transcription of this template in the absence of CTP positioned the elongation complex at position +25 (Fig. 3B, lane 1). Besides the expected G25 RNA product (the transcript and the corresponding stalled complex is termed G25 according to the type and the position of the last incorporated nucleotide), several smaller abortive RNA products were generated, which were removed by repeated washing of the elongation complex (lane 2), indicating that these products were already released from the ternary complex. The washed elongation complexes were still active since, after addition of unlabeled nucleotides, the stalled transcript disappeared almost completely, and the 121-nt runoff transcript was formed (lane 3).


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  		Fig. 3.   Formation of stalled elongation complexes. A, shown is a schematic drawing of the template pIC-31/30PRO-C25. The nucleotide sequence of the mutated region is shown from the transcription start site (position +1) to position +26. The lengths of the expected RNAs are indicated by arrows, and the promoter sequence is boxed. Immobilization of the template on magnetic beads by a streptavidin-biotin linkage is indicated by a filled circle. B, stalled elongation complexes were prepared by transcription in the absence of CTP as described under "Experimental Procedures" (lane 1). Template beads with stalled elongation complexes were washed twice with WAC buffer to remove unincorporated nucleotides and released or abortive RNAs and were resuspended in transcription buffer without MgCl2 (lane 2). G25 complexes were chased with 2 mM MgCl2 and 40 µM NTPs for 10 min (lane 3). The expected 25- and 121-nt RNAs are marked by arrows. RNA products were separated on a 17% denaturing polyacrylamide gel.

The TFIIS/RPSU Homologue Induces Cleavage Activity in the RNA Polymerase-- To analyze the function of the TFIIS/RPSU homologue, we incubated washed G25 elongation complexes with (Fig. 4, lane 13) or without (lane 3) the TFIIS/RPSU homologue. Addition of the TFIIS/RPSU homologue dramatically increased truncation of the nascent transcript (compare lanes 3 and 13). The truncation required Mg2+ (compare lanes 1 and 3 as well as lanes 11 and 13), which could be substituted by Mn2+ (compare lanes 3 and 5). Shortened RNAs remained in active ternary complexes, as nearly all of them could be elongated when unlabeled NTPs were added (lanes 4 and 14).


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  		Fig. 4.   Transcript cleavage by purified ternary elongation complexes. Washed ternary elongation complexes were resuspended in transcription buffer without MgCl2 and including the components indicated on top of each lane with the following final concentrations: 2 mM Mg2+, 2 mM Mn2+, 21 µM sodium pyrophosphate (Na-pyr), and 0.1 µM TFIIS/RPSU. After a 5-min incubation, the reactions were stopped by adding loading buffer or chased as indicated on top of the lanes. Chase reactions and transcriptional analysis were done as described in the legend to Fig. 3.

To address the question of whether truncation of the RNA in the absence of the TFIIS/RPSU homologue is mainly driven by pyrophosphorolysis due to low levels of endogenous pyrophosphate still bound to the washed elongation complexes, we added pyrophosphate in low concentrations (according to the low nucleotide concentrations used in the in vitro transcription reactions) to the G25 ternary complexes (Fig. 4, lane 9). Since addition of pyrophosphate had only a moderate influence on cleavage stimulation in the absence of the TFIIS/RPSU homologue (compare lanes 3 and 9), we assume that the RNA polymerase itself is able to drive the cleavage reaction.

To investigate the mechanism of the archaeal TFIIS/RPSU-induced cleavage in more detail and to get more information about RNA cleavage of the RNA polymerase itself, we have studied the time course of RNA truncation either in the absence or presence of the TFIIS/RPSU homologue. After a 1- or 2-min incubation of the washed elongation complexes in the presence of the TFIIS/RPSU homologue, the RNA in the G25 ternary complex was shortened in part to position -21 or -19, whereas the 15-nt elongation complex was barely detectable (Fig. 5, lanes 8 and 9). After a 15-min incubation, the RNA was completely truncated to position +15 (lane 11), whereas further incubation for 30 or 45 min did not result in further retraction of the RNA (lanes 12 and 13). Therefore, the time dependence of the cleavage pattern indicated that the TFIIS/RPSU homologue induced stepwise truncation of the RNA up to position +15. These 15-nt ternary complexes seem to be very stable since further truncation of the RNA did not occur. Cleavage activity in the absence of the TFIIS/RPSU homologue was dramatically reduced and was combined with the formation of some intermediate cleavage products and a slow accumulation of a 10-nt RNA (lanes 1-6). Addition of unlabeled nucleotides to the truncated RNAs revealed that all of these ternary complexes can continue elongation, except the 10-nt RNA accumulated in the absence of the TFIIS/RPSU homologue (lanes 7 and 14).


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  		Fig. 5.   Time course of transcript cleavage. Purified G25 complexes were retracted in the absence (lanes 1-7) or presence (lanes 8-14) of the TFIIS/RPSU homologue for the periods of time indicated on top of the lanes. Chase reactions were performed as described in the legend to Fig. 3. Inc., incubation.

TFIIS/RPSU Homologue-induced Cleavage Releases Dinucleotides-- The results obtained so far indicate that the archaeal TFIIS/RPSU homologue seems to have a function in archaeal transcription similar to that of TFIIS in eukaryotic pol II transcription. An additional feature of TFIIS-induced cleavage on stalled elongation complexes is the release of predominantly dinucleotides (19). Therefore, we have investigated the cleavage products released from retracted archaeal elongation complexes. For this analysis, washed G25 complexes were incubated for 45 min either in the absence or presence of the TFIIS/RPSU homologue. In the presence of the archaeal TFIIS/RPSU homologue, truncation of G25 ternary complexes was correlated with the release of dinucleotides (Fig. 6, lane 5). The 10-nt truncation of the 25- to the 15-nt RNA from the 3'-end in perfect dinucleotide increments should result in the release of the following five dinucleotides from the 3'-end: pApG, pUpA, pUpG, pApA, and pUpU (the labeled phosphates are underlined; see also Fig. 3A for the sequence). Using labeled pUpG and pApU as markers, we could demonstrate that the G25 complex liberates, in the presence of the TFIIS/RPSU homologue, the dinucleotides pUpG, pUpA, and most likely pUpU. Please note that the relative mobility of the dinucleotide pUpU is known to be slightly increased compared with that of pUpA in this gel system (19, 20). Furthermore, we assume that each of the bands corresponding to the expected pUpG and pUpA dinucleotides represents a mixture of pUpG/pGpU and pUpA/pApU, respectively. This assumption seems to be confirmed by the results of the calf intestinal alkaline phosphatase procedure (lane 7). Further treatment of the reaction including the 45-min incubation in the presence of the TFIIS/RPSU homologue was combined with a reduced electrophoretic mobility of the released dinucleotides, which indicates that the released dinucleotides contained a 5'-phosphate. Furthermore, we found, according to the distribution of the radioactive label and according to the behavior on the gel (20), most probably the three different dinucleotides GpU, ApU, and UpU instead of the predicted UpU, if retraction occurs in perfect dinucleotide increments.


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  		Fig. 6.   Cleavage products. Washed G25 ternary elongation complexes were retracted for 45 min either in the absence (lanes 1-4) or presence (lanes 5 and 7) of the TFIIS/RPSU homologue. To distinguish between released products and products that were still fixed in the elongation complex in the absence of the TFIIS/RPSU homologue (lane 1), we separated the templates including ternary complexes by the use of a magnet and analyzed the products still fixed in the elongation complexes (lane 3) as well as the released RNA products (lane 2) separately on a 28% denaturing polyacrylamide gel. A portion of the released products was further incubated for 15 min at 37 °C with 10 units of ribonuclease T1 before electrophoretic analysis (lane 4). For further analysis of the dinucleotides, a portion of the retracted elongation complex was incubated for 15 min at 37 °C with 10 units of calf intestinal phosphatase (CIP; lane 7). The position of the marker pUpG* is indicated by arrows. The presence of the markers pApU (lane 6) and ApU (lane 8) is indicated on top.

The most possible explanation for this finding is that the TFIIS/RPSU-induced truncation occurs in dinucleotide as well as mononucleotide increments because the removal of mononucleotides during the truncation process converted the predicted pUpG and pUpA dinucleotides into pGpU and pApU. The large amount of probably GpU could be caused most likely by a preference for releasing pGpU instead of pUpG.

Beside the dinucleotides, the G25 complexes also released a "non-chaseable" 10-nt RNA, which is the major product in the absence and a minor product in the presence of the archaeal TFIIS/RPSU homologue (Fig. 6, lanes 1 and 5; see also Fig. 5, lanes 7 and 14, for the chase experiment). Separation of bound and released RNAs revealed that the 10-nt RNA was released from the elongation complex (Fig. 6, lanes 1-3). Furthermore, ribonuclease T1 treatment increased the electrophoretic mobility, but did not change the amount of incorporated radioactivity. This indicates that the 10-nt RNA represents the sequence from the 3'-end and not from the 5'-end (Fig. 6, lane 4; see Fig. 3A sequence for details).

The TFIIS/RPSU Homologue Reduces the Amount of Arrested Complexes-- Since one major function of eukaryotic TFIIS is to aid RNA pol II at arrest sites, we have addressed the question of whether the archaeal TFIIS/RPSU homologue is able to prevent the RNA polymerase from going into arrest or to rescue arrested elongation complexes. For this analysis, we chased washed G25 elongation complexes (Fig. 7, lane 1) into a 424-nt runoff transcript either in the presence or absence of the archaeal TFIIS/RPSU homologue (lanes 2 and 3). In agreement with Fig. 3, most of the G25 elongation complexes could continue elongation after addition of nucleotides, but there were some elongation complexes that could not resume elongation. The amount of these complexes was very low; but most important, in the presence of the TFIIS/RPSU homologue, the amount of these non-chaseable complexes was reduced to about one-half (Fig. 7, compare lanes 2 and 3). We assume that most of these complexes that could not continue transcription were arrested because they were not released from the elongation complexes (data not shown).


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  		Fig. 7.   The TFIIS/RPSU homologue reduces the amount of arrested complexes. Purified G25 as well as A45 elongation complexes were prepared as described in the legend to Fig. 3 and under "Experimental Procedures" (lanes 1, 4, and 7). Chase reactions were done immediately with 40 µM NTPs for 6 min at 55 °C either in the absence or presence of the TFIIS/RPSU homologue (lanes 2, 3, 8, and 9) or after incubation for 2 h at 55 °C (lanes 5 and 6). The 25- and 45-nt RNAs as well as the runoff transcripts are marked by arrows. RNA products were separated on a 17% denaturing polyacrylamide gel.

Since longer incubation of the washed elongation complexes for 2 or 24 h at 0 °C or 2 h at 55 °C before the chase reaction did not increase the amount of arrested complexes (Fig. 7, lanes 5 and 6; and data not shown), we have analyzed pIC-31/30PRO-C45 as another template. This template is based on the promoter of pIC-31/30PRO-C25 with the difference that transcription in the absence of CTP positions the elongation complex at position +45 instead of +25. Washed A45 elongation complexes were prepared as described above and chased into the runoff transcript in the presence and absence of the TFIIS/RPSU homologue. As for the G25 complexes, the amount of arrested complexes was still very low, but in the presence of the TFIIS/RPSU homologue, the formation of arrested elongation complexes at position +45 was completely abolished (lanes 8 and 9). Please note that, in the presence of the TFIIS/RPSU homologue, the amount of runoff transcripts was also slightly increased in each case. Taken together, the reduction of the amount of arrested complexes at position +25 and the elimination of arrested complexes at position +45 are strong indications that the archaeal TFIIS/RPSU homologue is able to prevent or to rescue arrested elongation complexes and therefore is able to catalyze a similar function like TFIIS.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The potential of ternary elongation complexes to cleave nascent RNA from the 3'-end seems to be a common feature of eukaryotic as well as bacterial and viral RNA polymerases (reviewed in Refs. 21 and 22). Our data demonstrate that the cleavage activity is also present in the Methanococcus RNA polymerase and provide evidence that the ability to cleave nascent RNA is a common feature of archaeal ternary elongation complexes.

Meanwhile, it seems to be clear that this intrinsic cleavage activity resides in the RNA polymerase itself (23, 24), but that cleavage can be stimulated by additional factors. In E. coli, the two transcription factors GreA and GreB are responsible for stimulation of this activity (25, 26). External cleavage stimulation factors are also involved in eukaryotic pol I and II transcription. In the case of pol I, cleavage activity can be stimulated by an activity different from TFIIS (27, 28). For stimulation of cleavage, pol II requires the elongation factor TFIIS, which enables pol II to transcribe through a variety of blocks to transcription, including DNA-binding proteins and intrinsic arrest sites (reviewed in Refs. 22 and 29). TFIIS-induced cleavage on arrested (transcriptionally incompetent) complexes releases 7-14-nucleotide RNA fragments, whereas cleavage on stalled (transcriptionally competent) complexes releases predominantly dinucleotides (19, 30). The intrinsic cleavage activity of Methanococcus RNA polymerase can also be strongly stimulated by the TFIIS/RPSU homologue (Fig. 4). Please note that the different cleavage pattern in the absence of the TFIIS/RPSU homologue (Fig. 5) as well as the release of dinucleotides in the presence of the TFIIS/RPSU homologue (Fig. 6) are strong indications that cleavage without the TFIIS/RPSU homologue most likely originates from the RNA polymerase itself and not from trace contaminations of the TFIIS/RPSU homologue. Western blot analysis with antibodies against the recombinant TFIIS/RPSU homologue demonstrated that this protein is not a subunit of the RNA polymerase (Figs. 1 and 2). Furthermore, the presence of the archaeal TFIIS/RPSU homologue reduces the formation of arrested elongation complexes (Fig. 7). Taken together, the mechanism of archaeal RNA cleavage induction seems to be similar to the two-component system of pol II.

In the light of these results, it is interesting to discuss again the nature of the archaeal TFIIS/RPSU homologue. The hypothesis (mentioned in more detail in the Introduction) that the archaeal TFIIS/RPSU homologue is a subunit of the RNA polymerase was not supported by our experimental data. Furthermore, release of the corresponding subunits in eukaryotes during purification of the wild-type RNA polymerases has not been reported (13). Owing to these findings, this protein is much more likely to be an archaeal transcription factor similar to the eukaryotic elongation factor TFIIS than a subunit of the archaeal RNA polymerase. Furthermore, the archaeal TFIIS/RPSU homologue seems to be involved in transcriptional proofreading mechanisms similar to the eukaryotic TFIIS (31).2 To stress the similar function of the archaeal protein, we would like to suggest transcription factor S (TFS) as a name for the archaeal protein.

A similar function of archaeal TFS and eukaryotic TFIIS raises the question of the discrepancy of the molecular masses of these two proteins. Yeast TFIIS contains 309 amino acids, whereas Methanococcus TFS contains only 105 amino acids. Yeast TFIIS is composed of three structural domains (domains I-III) as defined by limited proteolytic digestion (32). Domain II (positions 131-240) and domain III (positions 241-309) are sufficient for biochemical activity in vitro and in vivo (33). Domain II interacts with the largest subunit of the RNA polymerase, and domain III contains a zinc ribbon that is involved in the mechanism of transcript cleavage (34). Domain III is well conserved in archaeal TFS: 37% of the amino acids are identical between domain III of yeast TFIIS and archaeal TFS (data not shown), indicating that the mechanism of cleavage seems to be very similar.

Interestingly, archaeal TFS shows also significant sequence similarity to the corresponding subunits of the RNA pol I, II, and III from Saccharomyces cerevisiae. Analysis of sequence similarities revealed 39% identity to C11, 32% to B12.6, and 31% to A12.2 (data not shown). A more detailed sequence analysis focusing on the carboxyl-terminal zinc-binding motifs between the archaeal protein and the corresponding yeast proteins revealed significant differences to B12.6 (Fig. 8). Including the cysteine residues, eight amino acids are conserved in all proteins (boxed), whereas four additional amino acids are conserved in all proteins except B12.6 (shaded). Interestingly, one of these four amino acids, Glu, which is not conserved in B12.6 (lowercase), together with the Asp residue, plays a critical role in cleavage mediated by TFIIS and C11 (discussed below), as demonstrated by mutational analysis (13, 35). The sequence Asp-Glu of eukaryotic TFIIS has been suggested to participate in metal binding and phosphoryl transfer within the pol II active site (35). The finding that the essential Asp-Glu dipeptide is conserved in all archaeal proteins (data not shown) but not in the B12.6 subunit suggests that the function of the archaeal protein is different from that of B12.6.


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  		Fig. 8.   Sequence alignment. Shown is the alignment of the carboxyl-terminal zinc-binding domains of TFS from Mc. thermolithotrophicus with RNA polymerase (RP) I A12.2, pol II B12.6, pol III C11, and TFIIS from S. cerevisiae. Identical amino acids in all sequences are boxed; amino acids conserved in all sequences but B12.6 are shaded. The amino acid that is essential for cleavage induction in TFIIS and C11 but that is not conserved in B12.6 is lowercase.

Functional analysis of this protein in yeast revealed that it is not essential for cell viability, but yeast pol II lacking subunit B12.6 (pol IIDelta ) showed an altered start site selection in vivo and in vitro (36, 37). Furthermore, pol IIDelta complexes undergo TFIIS-induced transcript cleavage, indicating that this protein is not directly involved in the mechanism of cleavage or cleavage induction (38). Taking these results together, as already indicated by the sequence comparison, B12.6 has clearly different functions than archaeal TFS, and this is a further confirmation that the archaeal protein does not represent a B12.6-like protein.

The finding that 39% of the amino acids are identical between TFS and C11 calls for a closer inspection of the mechanism of cleavage induction in these complexes. Highly purified pol III ternary complexes were found to possess a likely factor-independent cleavage activity (39). Analysis of the cleavage products indicated that truncation of the RNA is combined with the release of primarily dinucleotides and, to a lower extent, mononucleotides (39). Furthermore, cleavage activity in pol III ternary complexes strictly depends on the presence of the C11 subunit containing an intact Asp-Glu dipeptide (13). There is also no report of an external cleavage induction component in the pol III system. In contrast, cleavage in the ternary elongation complex of Methanococcus is also possible in the absence of the archaeal TFIIS/RPSU homologue (Fig. 5); and therefore, cleavage in the archaeal system does not absolutely require the Asp-Glu dipeptide of the TFIIS/RPSU homologue. We have also never observed the release of dinucleotides in the ternary elongation complex in the absence of the TFIIS/RPSU homologue. So we assume that the Asp-Glu dipeptide is necessary for the release of dinucleotides (Fig. 6) on stalled complexes; but in contrast to the pol III system, the archaeal ternary complex is also able to cleave the nascent RNA combined with the release of other nucleotides, but not of dinucleotides. Thus, the situation in the archaeal system clearly differs from the situation in the pol III system and resembles much more the pol II two-component system.

An interesting point that has to be discussed in more detail is the mode of interaction of TFS with the RNA polymerase in the archaeal two-component system. Since domain II of TFIIS, which is responsible for the binding of TFIIS to the RNA polymerase, is missing in TFS and domain III of TFIIS is unable to induce cleavage in the RNA polymerase without domain II, we assume that the mode of interaction of archaeal TFS with the RNA polymerase is different from that of TFIIS. Furthermore, experiments with recombinant C11 and pol III lacking C11 (pol IIIDelta ) indicate that the archaeal mode of interaction of TFS with RNA polymerase is probably still reminiscent of an artificial pol III system.

Recombinant C11 added to ternary elongation complexes with pol IIIDelta after purification of the ternary elongation complex by gel filtration chromatography is able to induce cleavage, but cleavage fails if recombinant C11 is added to transcription reactions before purification of the ternary complex (13). This observation indicates that recombinant C11 is not able to integrate into the right position of a pre-assembled pol III, but is nevertheless still able to stimulate cleavage in the ternary elongation complex (13). Furthermore, Riva and co-workers (13) could also demonstrate that C11 is important for termination of transcription, but in contrast to restoration of cleavage, addition of recombinant C11 to pol IIIDelta does not restore wild-type activity of termination. One possible explanation, also indicated by the results of the gel filtration chromatography, for this apparent discrepancy is that termination requires a stable integration of C11 into pol III.

Taken together, it is tempting to speculate that the results from this artificial situation, using recombinant C11 to induce cleavage in pol IIIDelta , and the high sequence similarity between C11 and TFS indicate that this observation of cleavage induction of recombinant C11 is reminiscent of Archaea. We assume that the role of recombinant C11 in transcription termination is an invention of pol III since it requires stable integration of this protein into the pol III complex; and furthermore, the archaeal protein does not seem to be involved in termination of archaeal transcription when using archaeal as well as eukaryotic pol III terminator sequences.2

    ACKNOWLEDGEMENTS

We thank Dagmar Haacks, Trevor Darcy, and E. P. Geiduschek for critical reading of the manuscript and Jutta Kock for technical assistance. We also thank Michael Thomm for helpful advice on the manuscript.

    FOOTNOTES

* This work was supported by Deutsche Forschungsgemeinschaft Grant HA 2612/1-1 and by Deutsche Forschungsgemeinschaft Grants TH 422/6-1 and TH 422/7-1 and grants from the Fonds der Chemischen Industrie (to Michael Thomm).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 49-431-880-1649; Fax: 49-431-880-2194; E-mail: whausner@ifam.uni-kiel.de.

2 W. Hausner and U. Lange, unpublished data.

    ABBREVIATIONS

The abbreviations used are: aTBP, archaeal TATA box-binding protein; TF, transcription factor; pol, RNA polymerase; PCR, polymerase chain reaction; bp, base pair(s); nt, nucleotide; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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