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• W2049732257 abstract "In living organisms, stable elongation complexes of RNA polymerase dissociate at specific template positions in a process of transcription termination. It has been suggested that the dissociation is not the immediate cause of termination but is preceded by catalytic inactivation of the elongation complex. In vitro reducing ionic strength can be used to stabilize very unstable and catalytically inactive complex at the point of termination; the previous biochemical characterization of this complex has led to important conclusions regarding termination mechanism. Here we analyze in detail the complexes formed between DNA template, nascent RNA, and Escherichia coli RNA polymerase during transcription through the tR2 terminator of bacteriophage λ. At low ionic strength, the majority of elongation complexes fall apart upon reaching the terminator. Released RNA and DNA efficiently rebind RNA polymerase (RNAP) and form binary RNAP·RNA and RNAP·DNA complexes, which are indistinguishable from binary complexes obtained by direct mixing of the purified nucleic acids and the enzyme. A small fraction of elongation complexes that reach termination point escapes dissociation because RNA polymerase has backtracked from the terminator to an upstream DNA position. Thus, transcription elongation to a terminator site produces no termination intermediates that withstand dissociation in the time scale appropriate for biochemical studies. In living organisms, stable elongation complexes of RNA polymerase dissociate at specific template positions in a process of transcription termination. It has been suggested that the dissociation is not the immediate cause of termination but is preceded by catalytic inactivation of the elongation complex. In vitro reducing ionic strength can be used to stabilize very unstable and catalytically inactive complex at the point of termination; the previous biochemical characterization of this complex has led to important conclusions regarding termination mechanism. Here we analyze in detail the complexes formed between DNA template, nascent RNA, and Escherichia coli RNA polymerase during transcription through the tR2 terminator of bacteriophage λ. At low ionic strength, the majority of elongation complexes fall apart upon reaching the terminator. Released RNA and DNA efficiently rebind RNA polymerase (RNAP) and form binary RNAP·RNA and RNAP·DNA complexes, which are indistinguishable from binary complexes obtained by direct mixing of the purified nucleic acids and the enzyme. A small fraction of elongation complexes that reach termination point escapes dissociation because RNA polymerase has backtracked from the terminator to an upstream DNA position. Thus, transcription elongation to a terminator site produces no termination intermediates that withstand dissociation in the time scale appropriate for biochemical studies. Structural stability is a trademark of ternary elongation complexes (ECs), 1The abbreviations used are: ECelongation complexRNAPRNA polymeraserNTPribonucleoside triphosphatentnucleotide(s)TBtranscription bufferTC/Nitermination complex isolated on Ni2+-NTA agaroseTC/Sttermination complex isolated on streptavidin-agaroseNTAnitrilotriacetic acid consisting of RNA polymerase (RNAP), template DNA, and newly synthesized transcript. Once formed on a promoter, EC can transcribe thousands of nucleotides and survive prolonged pausing and arrest within the transcribed genes (1.Uptain S.M. Kane C.M. Chamberlin M.J. Annu. Rev. Biochem. 1997; 66: 117-172Crossref PubMed Scopus (400) Google Scholar, 2.von Hippel P.H. Science. 1998; 281: 660-665Crossref PubMed Scopus (202) Google Scholar). The current view of EC structure, based on crystallographic and functional studies of eukaryotic, prokaryotic, and phage RNA polymerases, suggests that the enzyme holds the template with a clamp-like domain (3.Zhang G. Campbell E.A. Minakhin L. Richter C. Severinov K. Darst S.A. Cell. 1999; 98: 811-824Abstract Full Text Full Text PDF PubMed Scopus (675) Google Scholar, 4.Darst S.A. Curr. Opin. Struct. Biol. 2001; 11: 155-162Crossref PubMed Scopus (136) Google Scholar, 5.Landick R. Cell. 2001; 105: 567-570Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 6.Gnatt A.L. Cramer P. Fu J. Bushnell D.A. Kornberg R.D. Science. 2001; 292: 1876-1882Crossref PubMed Scopus (752) Google Scholar). This interaction does not interfere with advancement of RNAP along the template but makes the complex resistant to high salt concentration and DNA competitors (3.Zhang G. Campbell E.A. Minakhin L. Richter C. Severinov K. Darst S.A. Cell. 1999; 98: 811-824Abstract Full Text Full Text PDF PubMed Scopus (675) Google Scholar, 4.Darst S.A. Curr. Opin. Struct. Biol. 2001; 11: 155-162Crossref PubMed Scopus (136) Google Scholar, 5.Landick R. Cell. 2001; 105: 567-570Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 6.Gnatt A.L. Cramer P. Fu J. Bushnell D.A. Kornberg R.D. Science. 2001; 292: 1876-1882Crossref PubMed Scopus (752) Google Scholar). The minimal nucleic acid architecture that can support a stable bacterial EC and may allow the closure of the clamp around the DNA template, consists of 8–9 bp of an RNA:DNA hybrid at the 3′-end of the nascent RNA and a 9–12-bp DNA duplex located downstream from the hybrid (7.Nudler E. Avetissova E. Markovtsov V. Goldfarb A. Science. 1996; 273: 211-217Crossref PubMed Scopus (176) Google Scholar, 8.Sidorenkov I. Komissarova N. Kashlev M. Mol. Cell. 1998; 2: 55-64Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 9.Korzheva N. Mustaev A. Kozlov M. Malhotra A. Nikiforov V. Goldfarb A. Darst S.A. Science. 2000; 289: 619-625Crossref PubMed Scopus (338) Google Scholar). elongation complex RNA polymerase ribonucleoside triphosphate nucleotide(s) transcription buffer termination complex isolated on Ni2+-NTA agarose termination complex isolated on streptavidin-agarose nitrilotriacetic acid During transcription termination, processive RNA synthesis is interrupted by dissociating EC into RNAP, RNA, and DNA (1.Uptain S.M. Kane C.M. Chamberlin M.J. Annu. Rev. Biochem. 1997; 66: 117-172Crossref PubMed Scopus (400) Google Scholar, 2.von Hippel P.H. Science. 1998; 281: 660-665Crossref PubMed Scopus (202) Google Scholar). Termination in Escherichia coli occurs at two classes of specific DNA signals; one depends on action of termination factor Rho, and another requires only cis-acting element in DNA. The latter terminators are called intrinsic, or Rho-independent transcription terminators, and they cause spontaneous dissociation of EC (2.von Hippel P.H. Science. 1998; 281: 660-665Crossref PubMed Scopus (202) Google Scholar). Intrinsic terminators have a conserved structure consisting of a region of dyad symmetry followed by a stretch of 7–9 thymidine residues in nontemplate DNA strand (10.d'Aubenton Carafa Y. Brody E. Thermes C. J. Mol. Biol. 1990; 216: 835-858Crossref PubMed Scopus (290) Google Scholar). Transcription through this sequence results in formation of a stable hairpin in the RNA 7–9 nt upstream from the termination point followed by a run of U residues (2.von Hippel P.H. Science. 1998; 281: 660-665Crossref PubMed Scopus (202) Google Scholar). The mechanism of intrinsic termination has been studied for decades, and several models have been suggested to explain the disruption of ECs (for a review, see Ref. 11.Landick R. Science. 1999; 284: 598-599Crossref PubMed Scopus (27) Google Scholar). According to the established thermodynamic model, formation of the hairpin removes RNA from a putative single-strand RNA-binding site located on RNAP near the point where transcript branches from the RNA:DNA hybrid (12.Altmann C.R. Solow-Cordero D.E. Chamberlin M.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3784-3788Crossref PubMed Scopus (62) Google Scholar, 13.Wilson K.S. von Hippel P.H. J. Mol. Biol. 1994; 244: 36-51Crossref PubMed Scopus (51) Google Scholar, 14.Wilson K.S. von Hippel P.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8793-8797Crossref PubMed Scopus (195) Google Scholar, 15.Nudler E. Gusarov I. Avetissova E. Kozlov M. Goldfarb A. Science. 1998; 281: 424-428Crossref PubMed Scopus (74) Google Scholar, 16.Wilson K.S. Conant C.R. von Hippel P.H. J. Mol. Biol. 1999; 289: 1179-1194Crossref PubMed Scopus (27) Google Scholar). The model suggests that the hairpin formation also disrupts the 5′-proximal part of the RNA:DNA hybrid either directly, by displacing the RNA:DNA with the RNA:RNA pairing, or indirectly, by melting the hybrid in the region 3′ of the hairpin stem (2.von Hippel P.H. Science. 1998; 281: 660-665Crossref PubMed Scopus (202) Google Scholar, 16.Wilson K.S. Conant C.R. von Hippel P.H. J. Mol. Biol. 1999; 289: 1179-1194Crossref PubMed Scopus (27) Google Scholar, 17.Gusarov I. Nudler E. Mol. Cell. 1999; 3: 495-504Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). These events, in combination with low stability of oligo(U):dA hybrid at the terminator, provoke EC dissociation (18.Yager T.D. von Hippel P.H. Biochemistry. 1991; 30: 1097-1118Crossref PubMed Scopus (170) Google Scholar). Another, recently reported model considers the hairpin formation behind RNAP as the driving force for physical extraction of the 3′-end of the nascent RNA from the RNAP active center accompanied by hypertranslocation of RNAP along the DNA and by collapse of the transcription bubble (19.Yarnell W.S. Roberts J.W. Science. 1999; 284: 611-615Crossref PubMed Scopus (266) Google Scholar). Conformational changes in RNAP, caused by allosteric contacts between the hairpin and the enzyme, were also suggested to trigger termination (20.Arndt K.M. Chamberlin M.J. J. Mol. Biol. 1990; 213: 79-108Crossref PubMed Scopus (116) Google Scholar, 21.Toulokhonov I. Artsimovitch I. Landick R. Science. 2001; 292: 730-733Crossref PubMed Scopus (183) Google Scholar). In addition, direct destabilizing contacts between the termination hairpin and the protein clamp have been proposed, based on the results of protein/DNA and protein/RNA cross-linking experiments (15.Nudler E. Gusarov I. Avetissova E. Kozlov M. Goldfarb A. Science. 1998; 281: 424-428Crossref PubMed Scopus (74) Google Scholar, 17.Gusarov I. Nudler E. Mol. Cell. 1999; 3: 495-504Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). Depending on biochemical tools that the researchers used, each model proposed a distinct sequence of events leading to termination. However, it is not clear yet whether these events constitute true steps in the termination mechanism. Dissecting the early steps in the termination pathway would be extremely helpful for the understanding of the detailed mechanism of termination. Several studies suggest that RNAP may stop at a terminator before it dissociates from the DNA and RNA. Indeed, RNA hairpins similar to those found in the terminators often induce pausing of transcription at 10–12 nt downstream from their stems (22.Artsimovitch I. Landick R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7090-7095Crossref PubMed Scopus (324) Google Scholar). In the absence of hairpin, RNAP pauses at the end of 7–9-nt-long oligo(T) track (17.Gusarov I. Nudler E. Mol. Cell. 1999; 3: 495-504Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 19.Yarnell W.S. Roberts J.W. Science. 1999; 284: 611-615Crossref PubMed Scopus (266) Google Scholar). Thus, both the RNA hairpin and the oligo(T) track would be expected to cause pausing. Also, kinetic competition between dissociation and elongation at the termination point was shown to affect the efficiency of termination (23.McDowell J.C. Roberts J.W. Jin D.J. Gross C. Science. 1994; 266: 822-825Crossref PubMed Scopus (105) Google Scholar). Single-molecule light microscopy studies further suggested that pausing of E. coliRNAP for about 1 min precedes dissociation at the intrinsic terminator (24.Yin H. Artsimovitch I. Landick R. Gelles J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13124-13129Crossref PubMed Scopus (63) Google Scholar). The high rate of elongation and EC instability at the terminator hinder isolation and study of termination intermediates (17.Gusarov I. Nudler E. Mol. Cell. 1999; 3: 495-504Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 20.Arndt K.M. Chamberlin M.J. J. Mol. Biol. 1990; 213: 79-108Crossref PubMed Scopus (116) Google Scholar, 25.Nudler E. Kashlev M. Nikiforov V. Goldfarb A. Cell. 1995; 81: 351-357Abstract Full Text PDF PubMed Scopus (105) Google Scholar). Moreover, the study of the termination mechanism was complicated by the absence of a characterized biochemical system to isolate catalytically inactive intermediates before they dissociate. Reduction of the ionic strength below physiological level has been used as a tool to “freeze” such intermediates (17.Gusarov I. Nudler E. Mol. Cell. 1999; 3: 495-504Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 26.Fuller R.S. Platt T. Nucleic Acids Res. 1978; 5: 4613-4623PubMed Google Scholar). The complexes that remained stable at terminator at low salt conditions were immobilized in solid phase on nickel-NTA-agarose through histidine-tagged RNAP and were biochemically characterized (17.Gusarov I. Nudler E. Mol. Cell. 1999; 3: 495-504Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). Footprinting analysis of this complex showed that the normally opened transcription bubble had closed. These findings suggested that the closure of the transcriptional bubble represented an initial step in the termination pathway that accompanies catalytic inactivation of EC prior to its dissociation. Other evidence argued that in low ionic strength conditions, transcription termination and transcript release occurred, but nonspecific rebinding of the dissociated nucleic acids to the enzyme formed separate binary RNAP·RNA and RNAP·DNA post-termination complexes (27.Berlin V. Yanofsky C. J. Biol. Chem. 1983; 258: 1714-1719Abstract Full Text PDF PubMed Google Scholar). The immobilization through RNAP cannot separate ternary complexes from the binary adducts between the nucleic acids and the enzyme, imposing the problem of structural discrimination between a population of rebound complexes and an initial ternary intermediate in the termination process. Therefore, the conclusion that was made about the ternary nature of the termination complex rested heavily upon some assumptions that we believed needed to be fully verified. Here, we performed systematic analysis of the complexes formed between RNA, DNA, and E. coli RNAP in the course of termination in low salt conditions. To immobilize these complexes in solid phase, we utilized affinity tags introduced either to the protein or to the template DNA. We found no ternary termination complex. Instead, we found a small fraction of the polymerase in a ternary complex, which escaped dissociation by backtracking from the point of termination; all other polymerase complexes resulted from post-termination binding of the released template or transcript back to the enzyme. These findings are discussed in light of the current models of the mechanism of intrinsic transcription termination. The standard template for transcription was a 156-bp DNA fragment (DNA156) carrying the A1 promoter of bacteriophage T7 and the tR2 terminator of bacteriophage λ. It was a product of PCR amplification and was purified by PAGE. The template for transcription was 3′-biotinylated in the template DNA strand as follows. The PCR product contained a uniqueBamHI site 70 nt upstream from the +1-position of the T7A1 promoter. Following digestion with BamHI, the 3′-end was filled in by Klenow enzyme in the presence of 1 mm dGTP, dCTP, TTP, and 100 μm biotinylated dATP (Sigma) for 15 min at 25 °C. The biotinylated DNA156 was purified by phenol extraction, precipitated with ethanol, dissolved in transcription buffer (TB40 (numerical index indicates concentration of KCl in the buffer expressed in mm); 20 mmTris-HCl, pH 7.9, 40 mm KCl, 5 mmMgCl2, 1 mm β-mercaptoethanol), and used for EC11 formation in solution as described below. E. coli RNAP bearing a hexahistidine tag genetically fused to the carboxyl terminus of the β′ subunit was purified as described (28.Kashlev M. Nudler E. Severinov K. Borukhov S. Komissarova N. Goldfarb A. Methods Enzymol. 1996; 274: 326-334Crossref PubMed Scopus (78) Google Scholar) from extracts of the RL916 strain (obtained from Dr. R. Landick). Stable EC stalled at position +11 (EC11) was obtained by preincubating 2 pmol of the template with 2 pmol of the enzyme (unless indicated otherwise) in regular TB40 for 5 min at 37 °C and subsequently adding 10 μm trinucleotide RNA primer ApUpC and 20 μm rATP and rGTP for 5 min at 25 °C. The EC11 was next immobilized either on Ni2+-NTA-agarose or on sreptavidin-agarose beads (see below). 20 μl of Ni2+-NTA agarose (Qiagen) prewashed in TB40 was added to EC11 formed in solution as described above. After a 5-min incubation at 25 °C the immobilized EC11 was repeatedly (five times) washed with TB40. Then EC11 was either chased with the four rNTPs (100 μm rATP, rCTP, rGTP, and 10 μm of UTP unless indicated otherwise) or walked to a desired position by repeated alterations of washing (four washing steps with 1 ml of TB40 each) and incubation with a subset of rNTPs (10 μm each) for 5 min at 25 °C. The KCl concentration was adjusted to 500 mmduring the walking (TB500) to suppress pausing and arrest of RNAP. The transcripts were labeled by the incorporation of an appropriate [α-32P]rNTP (PerkinElmer Life Sciences; 40 μCi of the labeled rNTP (3000 Ci/mmol), 5 min at 25 °C)). The position of labeling was cytosine +12 unless stated otherwise. All reactions were stopped with an equal volume of gel loading buffer (50 mm EDTA, 10 m urea) unless indicated otherwise, and the products were separated on denaturing PAGE. 20 μl of streptavidin-agarose suspension (Sigma) were washed with TB40 and mixed with EC11 for 10 min at 25 °C with constant shaking. The DNA-immobilized complex was washed with 1 ml of TB40. All other manipulations were the same as described for transcription on Ni2+-NTA-agarose. Ten microliters of washed immobilized EC, containing either full-size or GreB-cleaved RNA, was incubated for 1 min (unless indicated otherwise) in TB containing 1 m KCl (TB1000). After 20 s of centrifugation (VWRbrand Mini Centrifuge; 6000 rpm, 2000 g), 5 μl of the supernatant was removed and combined with an equal volume of gel loading buffer (“S” fraction). The remaining pellet was washed with 1 ml of TB40, the volume of the sample was adjusted to 5 μl and combined with an equal volume of gel loading buffer (“Pw ” fraction). The “Total” fraction contained 10 μl of the nonfractionated immobilized EC combined with 10 μl of gel loading buffer. The DNA template for KMnO4 footprinting was labeled in 20 μl of immobilized EC11 with 10 units of T4 polynucleotide kinase (New England Biolabs) and 50 μCi of [γ-32P]dATP (7000 Ci/mmol; ICN Biomedicals, Inc.) for 10 min at 25 °C. After washing with TB40, the complex was used as starting material for the chase or walking reaction. The32P end-labeled complexes were treated with 1 mm KMnO4 at 25 °C for 1 min. The reaction was stopped by adding 1 μl of β-mercaptoethanol. The complexes were eluted from Ni2+-NTA-agarose with 50 mm EDTA, the DNA in the supernatants was precipitated in the presence of 4 μg of the carrier plasmid DNA, cleaved with 10% piperidine for 15 min at 90 °C, reprecipitated, lyophilized, dissolved in gel loading buffer, and separated on 6% denaturing PAGE. GreB protein was purified as described (29.Borukhov S. Goldfarb A. Methods Enzymol. 1996; 274: 315-326Crossref PubMed Scopus (24) Google Scholar). The cleavage reaction was performed in 10 μl of TB40 with 0.5 μg of GreB for 10 min at 25 °C. 0.5 pmol of RNAP core were immobilized on 3 μl of Ni2+-NTA-agarose beads and washed with TB40, and the volume was adjusted to 90 μl. In a separate tube, EC11 was obtained as described above, labeled in both the RNA and the DNA, and then chased with four rNTPs in TB1000 for 10 min. The total volume of the reaction was 40 μl. Following fractionation, 10 μl of the supernatant that contained labeled RNA64–65 (termination products of 64 and 65 nt long), RNA86 (run-off product), and DNA156 were combined with 90 μl of preimmobilized RNAP, making the final KCl concentration 140 mm. After 5 min of incubation at 25 °C with intense shaking, the mixture was washed with TB40. GreB cleavage and RNA extension were then performed as described above. To study potential intermediates formed in the course of transcription termination, we used a synthetic template carrying the well studied Rho-independent tR2 terminator of bacteriophage λ (14.Wilson K.S. von Hippel P.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8793-8797Crossref PubMed Scopus (195) Google Scholar,17.Gusarov I. Nudler E. Mol. Cell. 1999; 3: 495-504Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 19.Yarnell W.S. Roberts J.W. Science. 1999; 284: 611-615Crossref PubMed Scopus (266) Google Scholar, 25.Nudler E. Kashlev M. Nikiforov V. Goldfarb A. Cell. 1995; 81: 351-357Abstract Full Text PDF PubMed Scopus (105) Google Scholar). It was cloned downstream from the A1 promoter from bacteriophage T7 (Fig. 1A). The hexahistidine-tagged RNAP, which can be bound to Ni2+-NTA-agarose beads, was used in transcription reactions (28.Kashlev M. Nudler E. Severinov K. Borukhov S. Komissarova N. Goldfarb A. Methods Enzymol. 1996; 274: 326-334Crossref PubMed Scopus (78) Google Scholar, 30.Kashlev M. Martin E. Polyakov A. Severinov K. Nikiforov V. Goldfarb A. Gene (Amst.). 1993; 130: 9-14Crossref PubMed Scopus (111) Google Scholar). By immobilizing RNAP on beads, the enzyme can be walked in steps along the template by adding subsets of rNTPs alternated with washing of the beads. It also allowed the isolation of the polymerase from the DNA and RNA dissociated during termination and therefore could provide a way to isolate in the solid phase putative termination intermediates that retained stability. This approach has been exploited before for the study of the termination mechanism (17.Gusarov I. Nudler E. Mol. Cell. 1999; 3: 495-504Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 25.Nudler E. Kashlev M. Nikiforov V. Goldfarb A. Cell. 1995; 81: 351-357Abstract Full Text PDF PubMed Scopus (105) Google Scholar, 31.Uptain S.M. Chamberlin M.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13548-13553Crossref PubMed Scopus (41) Google Scholar). In the experiment of Fig. 1 B, RNA-labeled Ni2+-NTA-immobilized EC11 (the numerical index indicates the RNA length) was obtained as described under “Experimental Procedures.” The RNA in the complex was labeled by incorporation of [α-32P]rCTP to form EC12 followed by the chase to tR2 terminator with all four rNTPs. Concentration of KCl in the reaction was below physiological level (40 mm) to increase the lifetime of potential termination complex(es). Transcription in TB40 resulted in two RNA products of termination, the major one 64 nt long and the minor one 65 nt long (RNA64–65), and in run-off RNA 86 nt long (RNA86). To analyze dissociation of the complexes at the terminator, an aliquot of the chase reaction mixture was centrifuged, half of the supernatant was removed (lane 3), and the pellet was washed with TB40 (lane 2, Pw fraction). About 60% of the terminator-specific RNA64–65 was released, and 40% remained associated with RNAP. The RNAP that reached the end of the template (run-off complex) also was associated with the RNA (RNA86, lanes 2 and3). Stability of the RNA64–65 or RNA86 complexes with RNAP was much lower than that of the normal ECs (20.Arndt K.M. Chamberlin M.J. J. Mol. Biol. 1990; 213: 79-108Crossref PubMed Scopus (116) Google Scholar, 32.Komissarova N. Kashlev M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14699-14704Crossref PubMed Scopus (87) Google Scholar). These RNAs were released after only 1 min of incubation in 1 mKCl (TB1000) (lane 4) except for a small (∼10%) fraction of the RNA64–65 that remained bound to RNAP even after the wash with TB1000. We address the origin of this minor fraction below. To test whether DNA was also bound to RNAP after termination, we obtained EC12 with the labeled RNA and labeled template (Fig. 1 C) and then chased it with the four rNTPs in TB40. About 10% of the labeled DNA was released into the supernatant (lanes 2 and 3). After a subsequent wash with TB1000, only 20–30% of the labeled template remained bound to the Ni2+-NTA-agarose with the RNAP (see Fig. 2 B, bottom panel, lanes 5 and 6). Thus, more DNA than RNA64–65 was bound to polymerase in both high and low salt conditions. Greater retention of the DNA compared with RNA64–65 is most likely explained by a fraction of the stable ternary complexes becoming permanently arrested on the DNA template before reaching the terminator and the presence of the ternary complex containing the run-off RNA86, which has not dissociated in low salt (Fig. 1 C, lane 3). We shall define the fraction of EC64–65, which was retained on the beads after washing with TB40, as termination complex isolated on Ni2+-NTA agarose (TC/Ni). Next, we analyzed the structure of a transcriptional bubble in TC/Ni using potassium permanganate (KMnO4) footprinting, which detects unpaired thymidine residues in DNA (17.Gusarov I. Nudler E. Mol. Cell. 1999; 3: 495-504Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 33.Komissarova N. Kashlev M. J. Biol. Chem. 1997; 272: 15329-15338Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 34.Komissarova N. Kashlev M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1755-1760Crossref PubMed Scopus (300) Google Scholar). Because of the very low stability of TC/Ni, it was crucial to test whether the complex would withstand the standard footprinting conditions (1 mmKMnO4 for 5 min (17.Gusarov I. Nudler E. Mol. Cell. 1999; 3: 495-504Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 33.Komissarova N. Kashlev M. J. Biol. Chem. 1997; 272: 15329-15338Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 34.Komissarova N. Kashlev M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1755-1760Crossref PubMed Scopus (300) Google Scholar)). Under these conditions, EC57, a regular EC, which was used as a control, was stable (Fig. 2 A, lanes 3 and 4). TC/Ni remained intact in TB40 (lanes 5 and6), but it released most of the RNA after 5 min following incubation with 1 mm KMnO4 in TB40 (lanes 7 and 8). The half-life of TC/Ni in these conditions (1 mm KMnO4) was less than 2–3 min (data not shown); therefore, more than 80% of the complex would dissociate under standard footprinting conditions. Dissociation of TC/Ni would lead to significant attenuation of the signal from the unpaired thymidines relative to that of regular stable ECs. However, after 1 min of incubation with 1 mmKMnO4, TC/Ni remained intact (lanes 9and 10). We chose this condition to probe for the transcriptional bubble. Fig. 2 B shows KMnO4 footprint of the nontemplate DNA strand in TC/Ni and in a series of ECs halted in the vicinity of the termination site. As expected, in EC46 the bubble was located at a position corresponding to the 3′-end of the RNA (lane 2). Elongation of the RNA to 57 nt was accompanied by forward progression of the bubble (lane 3). Extension of the RNA by another 5 nt (to form EC62) halts RNAP after the fifth nucleotide of the oligo(T) stretch of the tR2 terminator. In this position, RNAP becomes arrested and slides backwards along the DNA and the RNA (33.Komissarova N. Kashlev M. J. Biol. Chem. 1997; 272: 15329-15338Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 34.Komissarova N. Kashlev M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1755-1760Crossref PubMed Scopus (300) Google Scholar), causing rearrangement of the bubble and its retreat to the more upstream position (lane 4). The arrest of EC62 is reversible (data not shown), and the RNAP with the DNA bubble returns infrequently to the original location at the 3′-end of the RNA. Thus, the two bubble positions overlap in EC64, creating the impression of the unusually large size of the DNA opening in this complex. Lane 5 demonstrates the crucial result of this experiment. Although the amount of TC/Ni withdrawn for the footprinting reaction was the same as the amount of the other complexes (Fig. 2 B, top panel), no DNA bubble was detected in the complex as revealed by the resistance of thymidines in TC/Ni to KMnO4 (Fig. 2 B, lane 5). Lanes 5 and 6 of Fig. 2 B show that washing with 1 m KCl removed most of the RNA from TC/Ni, but the small fraction that remained generated the same footprint as the total TC/Ni. As we demonstrate below, this footprint originated from the minor salt-resistant fraction of ternary termination complexes described above (see also Fig. 1 B,lane 4). Thus, apart from this minor fraction, there was no transcription bubble detected in the majority of the DNA located in TC/Ni. Normally, the DNA duplex is melted inside the RNAP ternary complex (33.Komissarova N. Kashlev M. J. Biol. Chem. 1997; 272: 15329-15338Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 34.Komissarova N. Kashlev M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1755-1760Crossref PubMed Scopus (300) Google Scholar, 35.Zaychikov E. Denissova L. Heumann H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1739-1743Crossref PubMed Scopus (82) Google Scholar). Thus, it is extremely unusual if the TC/Ni represents a ternary complex that lacks the bubble. Previous studies had shown that low salt could stimulate formation of binary RNAP·RNA and RNAP·DNA complexes resulting from reassociation of RNAP with the nucleic acids released in the course of termination (27.Berlin V. Yanofsky C. J. Biol. Chem. 1983; 258: 1714-1719Abstract Full Text PDF PubMed Google Scholar). Therefore, we pe" @default.
• W2049732257 created "2016-06-24" @default.
• W2049732257 creator A5007385141 @default.
• W2049732257 creator A5086553695 @default.
• W2049732257 date "2002-04-01" @default.
• W2049732257 modified "2023-10-16" @default.
• W2049732257 title "Transcription Termination: Primary Intermediates and Secondary Adducts" @default.
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