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• W2054915693 abstract "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. 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. archaeal TATA box-binding protein transcription factor RNA polymerase polymerase chain reaction base pair(s) nucleotide N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine 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.Thomm M. FEMS Microbiol. Rev. 1996; 18: 159-171Crossref PubMed Google Scholar, 2.Reeve J.N. Sandman K. Daniels C.J. Cell. 1997; 89: 999-1002Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 3.Bell S.D. Jackson S.P. Trends Microbiol. 1998; 6: 222-228Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). These two factors together with RNA polymerase are necessary and sufficient for initiation of in vitro transcription at some promoters (4.Hausner W. Wettach J. Hethke C. Thomm M. J. Biol. Chem. 1996; 271: 30144-30148Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 5.Qureshi S.A. Bell S.D Jackson S.P. EMBO J. 1997; 16: 2927-2936Crossref PubMed Scopus (120) Google Scholar). 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 fulgidusindicated that there are, with the exception of a putative α-subunit of TFIIE, no further eukaryote-like initiation factors corresponding to TFIIA, TFIIF, or TFIIH (6.Bult C.J. White O. Olsen G.J. Zhou L. Fleischmann R.D. Sutton G.G. Blake J.A. FitzGerald L.M. Clayton R.A. Gocayne J.D. Kerlavage A.R. Dougherty B.A. Tomb J.-F. Adams M.D. Reich C.I. Overbeek R. Kirkness E.F. Weinstock K.G. Merrick J.M. Glodek A. Scott J.L. Geoghagen N.S.M. Weidman J.F. Fuhrmann J.L. Nguyen D. Utterback T.R. Kelley J.M. Peterson J.D. Sadow P.W. Hanna M.C. Cotton M.D. Roberts K.M. Hurst M.A. Kaine B.P. Borodovsky M. Klenk H.-P. Fraser C.M. Smith H.O. Woese C.R. Venter J.C. Science. 1996; 273: 1058-1073Crossref PubMed Scopus (2284) Google Scholar, 7.Smith D.R. Doucette-Stamm L.A. Deloughery C. Lee H. Dubois J. Aldredge T. Bashirzadeh R. Blakely D. Cook R. Gilbert K. Harrison D. Hoang L. Keagle P. Lumm W. Pothier B. Qiu D. Spadafora R. Vicaire R. Wang Y. Wierzbowski J. Gibson R. Jiwani N. Caruso A. Bush D. Safer H. Patwell D. Prabhakar S. McDougall S. Shimer G. Goyal A. Pietrokovski S. Church G.M. Daniels C.J. Mao J. Rice P. Nölling J. Reeve J.N. J. Bacteriol. 1997; 179: 7135-7155Crossref PubMed Scopus (1037) Google Scholar, 8.Kawarabayasi Y. Sawada M. Horikoshi H. Haikawa Y. Hino Y. Yamamoto S. Sekine M. Baba S. Kosugi H. Hosoyama A. Nagai Y. Sakai M. Ogura K. Otsuka R. Nakazawa H. Takamiya M. Ohfuku Y. Funahashi T. Tanaka T. Kudoh Y. Yamazaki J. Kushida N. Oguchi A. Aoki K. Yoshizawa T. Nakamura Y. Robb F.T. Horikoshi K. Masuchi Y. Shizuya H. Kikuchi H. DNA Res. 1998; 5: 55-76Crossref PubMed Scopus (553) Google Scholar, 9.Klink H.P. Clayton R.A. Tomb J.F. White O. Nelson K.E. Ketchum K.A. Dodson R.J. Gwinn M. Hickey E.K. Peterson J.D. Richardson D.L. Kerlavage A.R. Graham D.E. Kyrpides N.C. Fleischmann R.D. Quackenbush J. Lee N.H. Sutton G.G. Gill S. Kirkness E.F. Dougherty B.A. McKenney K. Adams M.D. Loftus B. Peterson S. Reich C.I. McNeil L.K. Badger J.H. Glodek A. Zhou L. Overbeek R. Gocayne J.D. Weidman J.F. McDonald L. Utterback T. Cotton M.D. Spriggs T. Artiach P. Kaine B.P. Sykes S.M. Sadow P.W. D'Andrea K.P. Bowman C. Fujii C. Garland S.A. Mason T.M. Olsen G.J. Fraser C.M. Smith H.O. Woese C.R. Venter J.C. Nature. 1997; 390: 364-370Crossref PubMed Scopus (1201) Google Scholar). 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.Langer D. Zillig W. Nucleic Acids Res. 1993; 21: 2251Crossref PubMed Scopus (32) Google Scholar). 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.Bell S.D. Jackson S.P. Trends Microbiol. 1998; 6: 222-228Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar,11.Kaine B.P. Mehr I.J. Woese C.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3854-3856Crossref PubMed Scopus (37) Google Scholar, 12.Langer D. Hain J. Thuriaux P. Zillig W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5768-5772Crossref PubMed Scopus (262) Google Scholar, 13.Chédin S. Riva M. Schultz P. Sentenac A. Carles C. Genes Dev. 1998; 12: 3857-3871Crossref PubMed Scopus (151) Google Scholar, 14.Soppa J. Mol. Microbiol. 1999; 31: 1295-1305Crossref PubMed Scopus (113) Google Scholar). 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 polymerasesubunit) 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. [γ-32P]ATP and [α-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. The plasmid pIC-31/30PRO-C25 used in this study is based on the tRNAValpromoter of Methanococcus vannielii (15.Hausner W. Frey G. Thomm M. J. Mol. Biol. 1991; 222: 495-508Crossref PubMed Scopus (85) Google Scholar). The cytosine residues in the region from positions +2 to +25 were substituted by other bases (see Fig. 3 A) 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 withHindIII, 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 theHindIII 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.Figure 1The TFIIS/RPSU homologue is not necessary forin 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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 andEcoRI and inserted between the SapI andEcoRI restriction sites of plasmid pIC-31/30PRO-C25 to generate pIC-31/30PRO-C45. RNA polymerase fromMc. thermolithotrophicus was purified as described previously (16.Hausner W. Thomm M. J. Biol. Chem. 1993; 268: 24047-24052Abstract Full Text PDF PubMed Google Scholar). 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 A 600 = 0.8 at 25 °C. Expression of the proteins was induced by addition of 1 mmisopropyl-1-thio-β-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. The coding region of TFB (GenBankTM/EBI accession number AJ271467) was subcloned using PCR amplification to generate the coding region with anNdeI restriction site at the 5′-end of the sequence. The amplified insert was then cloned between the NdeI andEcoRV restriction sites of the pET17b expression vector to generate pTFBMth.17. BL21(DE3) cells containing the pTFBMth.17 plasmid were grown to A 600 = 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. 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 mmNaCl, 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. 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 was performed as described previously (17.Hausner W. Thomm M. J. Biol. Chem. 1995; 270: 17649-17651Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). In vitrotranscription 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[α-32P]UTP (370 Bq/pmol) in transcription buffer (20 mm Tris, pH 8.5 at 20 °C, 2 mmMgCl2, 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.Hausner W. Thomm M. J. Biol. Chem. 1993; 268: 24047-24052Abstract Full Text PDF PubMed Google Scholar). 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[α-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 mmdithiothreitol, 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. RNA size markers were formed by in vitrotranscription 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 [γ-32P]ATP (9000 Bq/μl) in 50 mm Tris, pH 7.6, 10 mm MgCl2, and 5 mmdithiothreitol. The reactions were diluted in transcription and loading buffers. 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.1 A. To address the question of whether this protein is a component of the Methanococcus in vitro transcription system (18.Frey G. Thomm M. Brudigam B. Gohl H.P. Hausner W. Nucleic Acids Res. 1990; 18: 1361-1367Crossref PubMed Scopus (41) Google Scholar), 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.1 B, 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. 1 C, 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). The RNA polymerase used for in vitrotranscription is purified by four chromatographic steps (16.Hausner W. Thomm M. J. Biol. Chem. 1993; 268: 24047-24052Abstract Full Text PDF PubMed Google Scholar). 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.2 A). 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. 2 A). In contrast, the bulk of the TFIIS/RPSU homologue eluted in fractions 33–37, as shown by Western blot analysis (Fig. 2 B, 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. 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 tRNAValpromoter 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. 3 B,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). 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 and3 as well as lanes 11 and 13), which could be substituted by Mn2+ (compare lanes 3and 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). 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 and9). 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). 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.Izban M.G. Luse D.S. J. Biol. Chem. 1993; 268: 12864-12873Abstract Full Text PDF PubMed Google Scholar). 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, andpUpU (the labeled phosphates are underlined; see also Fig. 3 A 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.Izban M.G. Luse D.S. J. Biol. Chem. 1993; 268: 12864-12873Abstract Full Text PDF PubMed Google Scholar, 20.Bobkova E.V. Hall B.D. J. Biol. Chem. 1997; 272: 22832-22839Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). 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.Bobkova E.V. Hall B.D. J. Biol. Chem. 1997; 272: 22832-22839Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), most probably the three different dinucleotides GpU, ApU, and UpU instead of the predicted UpU, if retraction occurs in perfect dinucleotide increments. 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.3 A sequence for details). 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 and3). 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). 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. The potential of ternary elongation complexes to cleave nascent" @default.
• W2054915693 created "2016-06-24" @default.
• W2054915693 creator A5002395814 @default.
• W2054915693 creator A5059661064 @default.
• W2054915693 creator A5062204236 @default.
• W2054915693 date "2000-04-01" @default.
• W2054915693 modified "2023-10-17" @default.
• W2054915693 title "Transcription Factor S, a Cleavage Induction Factor of the Archaeal RNA Polymerase" @default.
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