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Originally published In Press as doi:10.1074/jbc.M701483200 on May 14, 2007

J. Biol. Chem., Vol. 282, Issue 26, 19020-19028, June 29, 2007
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Direct Versus Limited-step Reconstitution Reveals Key Features of an RNA Hairpin-stabilized Paused Transcription Complex*

Scotty Kyzer{ddagger}1, Kook Sun Ha{ddagger}, Robert Landick§2, and Murali Palangat§3

From the Departments of {ddagger}Biomolecular Chemistry and §Biochemistry, University of Wisconsin, Madison, Wisconsin 53706

Received for publication, February 20, 2007 , and in revised form, May 10, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We have identified minimal nucleic acid scaffolds capable of reconstituting hairpin-stabilized paused transcription complexes when incubated with RNAP either directly or in a limited step reconstitution assay. Direct reconstitution was achieved using a 29-nucleotide (nt) RNA whose 3'-proximal 9–10 nt pair to template DNA within an 11-nt noncomplementary bubble of a 39-bp duplex DNA; the 5'-proximal 18 nt of RNA forms the his pause RNA hairpin. Limited-step reconstitution was achieved on the same DNAs using a 27-nt RNA that can be 3'-labeled during reconstitution and then extended 2 nt past the pause site to assay transcriptional pausing. Paused complexes formed by either method recapitulated key features of a promoter-initiated, hairpin-stabilized paused complex, including a slow rate of pause escape, resistance to transcript cleavage and pyrophosphorolysis, and enhancement of pausing by the elongation factor NusA. These findings establish that RNA upstream from the pause hairpin and pyrophosphate are not essential for pausing and for NusA action. Reconstitution of the his paused transcription complex provides a valuable tool for future studies of protein-nucleic interactions involved in transcriptional pausing.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Synthesis of mRNA by DNA-dependent RNA polymerase (RNAP)4 is regulated during chain elongation by extrinsic elongation factors and by intrinsic signals encoded in the DNA and RNA, such as pause signals. At pause sites, a fraction of the elongating complex (EC) is temporarily halted (delayed in the addition of the next nucleotide). At the his pause site, which occurs in the leader region of the histidine biosynthetic operon of Escherichia coli, RNAP pauses until a translating ribosome is able to direct transcription readthrough or termination in response to the level of charged histidyl-tRNA (1, 2). This pause signal is multipartite and involves several nucleic acid segments that contact RNAP in the paused transcription complex (PTC): (i) a pause RNA hairpin, (ii) spacer RNA between the hairpin and the hybrid, (iii) the RNA:DNA hybrid, and (iv) duplex DNA downstream of the pause site (3–8). The pause mechanism remains incompletely understood, in part due to the difficulty of assembling pure populations of PTCs in vitro using DNA, RNA, and RNAP.

During active transcription, PTCs form from an intermediate in the nucleotide addition cycle, which begins by translocation to shift the RNA 3' nt from the substrate site (called the A or i + 1 site) in a pretranslocated EC to the product site (P or i site) in a posttranslocated EC with concomitant forward translocation of RNAP along the DNA chains. The rest of the cycle consists of NTP binding, catalysis, and pyrophosphate release to generate a new pretranslocated EC. PTCs appear to form when interactions of RNAP with the pause signal components in the pretranslocated EC create a change in the active site that transiently delays nucleotide addition without affecting the translocation register (8).

Three classes of pause signals have been recognized based on different mechanisms of prolonging the pause. Class I pauses are stabilized by the interaction of a nascent RNA hairpin with the RNAP flap and clamp domains (9). Class II pauses are stabilized by RNAP backtracking along the RNA and DNA chains (9–11). Class III pauses are stabilized by the interaction of dissociable factors with RNAP (e.g. {sigma}70 with RNAP shortly after promoter escape) (12). The his pause (Class I) is stabilized by the interaction of a 5-bp stem, 8-nt loop pause RNA hairpin that forms 11 nt from the paused transcript 3' end and contributes a factor of ~10 of an overall 100–1000-fold decrease in the nucleotide addition rate. In addition, the duration of the his pause can be increased ~2–4-fold by the elongation factor NusA, which interacts with the pause hairpin and with RNAP (7, 9). The his PTC remains pretranslocated but is resistant to pyrophosphorolysis, which ordinarily occurs rapidly in pretranslocated complexes (8). This suggests that the active site of the his PTC is altered such that catalysis is blocked.

Methods that allow direct reconstitution of PTCs without nucleotide addition or with a limited number of nucleotide addition steps would avoid the heterogeneity in ECs that occurs when transcription is initiated from a promoter and instead allow formation of homogeneous populations of PTCs. Daube and von Hippel (13) first demonstrated reconstitution of active ECs in vitro by incubating RNAP with a preformed RNA/DNA scaffold that forms a synthetic transcription bubble. ECs have been reconstituted in vitro to study active, nonpaused ECs with many of the same characteristics as elongation complexes initiated from a promoter (14, 15). However, the kinetic properties of reconstituted ECs have not been characterized. It is not known if reconstituted ECs are kinetically homogeneous and if PTCs can be reconstituted directly without prior nucleotide addition. In this report, we describe two methods to reconstitute PTCs: direct reconstitution and limited step reconstitution assay (LSRA). We characterized the kinetic species present in the population of reconstituted complexes by assaying nucleotide addition rates and the properties of reconstituted PTCs (rPTCs) compared with promoter-initiated his PTCs. We use rPTCs to address several fundamental questions about contributions of nucleic acid architecture to pausing, the ability of rPTCs to respond to protein elongation factors, and the possible role of PPi in pausing.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—NTPs, HisTrap HP, and Superdex 200 were from GE Healthcare (Piscataway, NJ), RNA oligonucleotides were from Dharmacon (Lafayette, CO), and DNA oligonucleotides were from Gene Link (Hawthorne, NY) or IDT (Corvalville, IA). All DNA and RNA oligonucleotides used in this study are listed in Table 1 and were purified by denaturing PAGE before use.


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  		TABLE 1
Oligonucleotides used in this study

 
Proteins—Chromosomal core E. coli RNAP was purified as described previously (16). GreB was a generous gift from Dr. Irina Artsimovitch. NusA was purified from BL21 {lambda}DE3 cells containing a NusA overexpression plasmid (pNG5) by a two-step purification protocol using HisTrap HP affinity chromatography followed by gel filtration on Superdex 200.5

PTC Reconstitution and Gel Shift Assay—The nucleic acid scaffolds for reconstituting PTCs were assembled in reconstitution buffer (RB; 10 mM Tris·HCl, pH 7.9, 40 mM KCl, 5 mM MgCl2) by heating 5'-32P-labeled RNA 29 (Table 1, 4865, 5.0 µM), tDNA (Table 1, 4891, 2.5 µM), and ntDNA (Table 1, 5051, 12.5 µM) containing an 11-nt noncomplementary region (bubble) to 75 °C for 2 min, rapidly cooling to 45 °C, and then cooling to room temperature in 2 °C/2-min steps as described previously (14). Reconstitution of paused and nonpaused ECs was performed by incubating core E. coli RNAP (2.5 µM) with the nucleic acid scaffold in RB for 10 min at 37 °C as described (13). When fully complementary DNA strands were used, RNA and tDNA were preannealed and incubated with RNAP at room temperature for 10 min followed by incubation with ntDNA for an additional 10 min at 37 °C. PTC reconstitution was monitored by gel shift assay by electrophoresing an aliquot of rPTCs on a 4–15% native polyacrylamide Phast gel (GE Healthcare). The gel was exposed to a PhosphorImager screen and quantitated using ImageQuant software (GE Healthcare).

In Vitro Nucleotide Addition Assays—Pause assays using promoter initiated ECs (template DNA from pIA171) were performed as described previously (6). The nucleotide addition assay was performed using rPTCs with 5'-32P-labeled RNA oligonucleotide. The rPTCs were diluted in RB to contain 25–50 nM rPTCs and elongated with GTP (10 µM) and ATP (100 µM) at 37 °C. Samples were removed at predetermined times, quenched with an equal volume of 2x loading dye (8 M urea, 50 mM EDTA, 90 mM Tris-borate buffer, pH 8.3, 0.02% bromphenol blue, and 0.02% xylene cyanol), and analyzed by denaturing 15% polyacrylamide gel electrophoresis. For experiments in Fig. 2, ECs were formed using 5'-32P-labeled RNA27 or RNA28 and elongating them in the presence of CTP and UTP to make U29 rPTCs in a limited-step transcription assay (5). When NusA was added, rPTCs were preincubated with NusA for 5 min at 37 °C followed by the addition of NTPs. Aliquots were removed at predetermined time intervals, and samples were processed as described above.

LSRA—C28 ECs were formed using LSRA by first reconstituting G27 complexes as above using unlabeled RNA27, tDNA, and ntDNA (4867, 4891, and 5069, respectively; Table 1) and 3'-labeling the transcript by including [{alpha}-32P]CTP (specific activity 3000 Ci/mmol; 2 µM) in the reconstitution reaction (Figs. 3, 5, and 6). The labeled C28 ECs were then diluted in transcription buffer (25 mM HEPES-KOH, pH 8.0, 130 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol, 0.15 mM EDTA, 5% glycerol, and 25 µg of acetylated bovine serum albumin/ml) to 25–50 nM and elongated in the presence of GTP (10 µM) and UTP (100 µM). LSRA offers many advantages for characterization of pausing. Only active ECs are visualized, and the rates of pause formation, pause escape, and pause bypass all can be measured at a wide range of substrate concentrations.

Rapid Quench-flow Transcription Reactions—Rapid quench-flow experiments were performed on a KinTek rapid quenched-flow apparatus. 20 µl of diluted C28 ECs were injected into one of the sample loops, and 20 µl of NTPs (20 µM GTP and 2 mM UTP) in transcription buffer was injected into the other loop. Reactions were performed for predetermined times at 37 °C, and samples were quenched with 2 M HCl and immediately neutralized to pH 8.0 with 3 M Tris. The RNA was phenol-extracted, ethanol-precipitated, suspended in 1x loading dye, and fractionated on a denaturing 17.5% polyacrylamide gel. Each time point represents a separate reaction on the quenched-flow apparatus.

Pyrophosphorolysis and GreB Cleavage Assay—Pyrophosphorolysis was performed on rPTC29 and rEC14 by incubating rPTCs/rECs (~100 nM) with PPi (1 mM) in RB at 37 °C. Aliquots were removed at the indicated times and quenched with an equal volume of 2x loading dye and analyzed by denaturing 15% polyacrylamide gel electrophoresis. GreB-mediated transcript cleavage of rPTC29 was performed by incubating rPTC29 (~100 nM) with GreB (4 µM) in RB at 37 °C. At the indicated times, aliquots were removed, and samples were processed as for the pyrophosphorolysis assay.

Data Quantitation and Analysis—Gels were exposed to storage phosphor screens and quantitated using a Typhoon PhosphorImager and ImageQuant software (GE Healthcare). The 29-mer pause RNA (U29) present in each lane was quantitated as a fraction of the total RNA in each lane (U29-A37 for direct reconstitution (Fig. 1); C28-G31 for LSRA (all other figures)) and corrected for the fraction remaining in the chase lane. The rate of pause escape was determined by nonlinear regression of [U29] versus time with either single or double exponential decay (see "Results").


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
PTCs Can Be Directly Reconstituted in Vitro Using Hairpin-containing RNA—To reconstitute the his PTC, we first tested a minimal scaffold patterned after the known pause signal components (pause hairpin, 2-nt spacer, 9-bp RNA:DNA hybrid, ~15-bp of downstream DNA; Fig. 1A) (3, 4, 7, 17). The 29-mer RNA contained the 18-nt his pause RNA hairpin but lacked RNA upstream of the hairpin. EC reconstitution was performed by incubating RNAP with a preformed RNA/DNA scaffold containing an 11-nt bubble (Fig. 1A; see "Experimental Procedures") (13). The resulting ECs were tested by electrophoretic shift of the 32P-labeled nucleic acid scaffold on a native polyacrylamide gel and also for ability to elongate the RNA in the presence of GTP and ATP to yield an RNA product 8 nt longer (Fig. 1, A and B). Under these conditions, ~40% of the labeled DNA/RNA scaffold reconstituted with RNAP to form an EC that migrated slowly on a native polyacrylamide gel (Fig. 1B, compare lanes 1 and 2). The RNA not shifted was either not incorporated into an EC or failed to anneal to the DNA strands. A fully complementary nontemplate strand yielded slightly lower efficiency (30%) (Fig. 1B, compare lanes 2 and 3). The amount of rPTCs able to extend the labeled RNA in the presence of GTP and ATP was similar to the amount that shifted in the gel shift assay when either noncomplementary or fully complementary nontemplate strands were used (Fig. 1B, compare lanes 2 and 5 with lanes 3 and 6). Since the amount of RNA that does not reconstitute into PTCs can be corrected for mathematically, we avoided perturbing the PTCs with techniques to remove the unbound scaffold.


Figure 1
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  		FIGURE 1.
Direct reconstitution of hairpin-containing rPTCs and pause escape. A, nucleic acid scaffold architecture and PTC reconstitution. PTCs were reconstituted using 5'-end-labeled hairpin RNA (RNA 29, 4865), tDNA (4891), and ntDNA (5051) strand oligonucleotides (Table 1) (see "Experimental Procedures"). RNA29 extended by RNAP is shown in lowercase type. B, PTC formation and transcript elongation. PTCs reconstituted as in A using either noncomplementary ntDNA (5051; lane 2) or fully complementary ntDNA strands (5041; lane 3) were separated on a native 4–12% polyacrylamide Phast gel (left). The positions of the free RNA (lane 1) and the shifted PTCs are indicated. The rPTCs (noncomplementary bubble (lane 5) and fully complementary scaffold (lane 6)) were elongated in the presence of ATP (100 µM) and GTP (10 µM), and the RNA products were separated on a denaturing polyacrylamide (15%) gel (right). Percentage shifted or elongated is indicated below each lane. The positions of the 29-nt RNA (lane 4) and other RNA products are indicated. C, kinetic analysis of reconstituted PTCs containing 5'-labeled hairpin RNA. rPTCs reconstituted as in A were elongated in the presence of ATP (100 µM) and GTP (10 µM), aliquots were removed at the indicated times and mixed with 2x loading dye, and RNA products were separated on 15% denaturing polyacrylamide gel. The lane marked C denotes the chase reaction in which [GTP] was adjusted to 5 mM after the time course, and incubation continued for an additional 5 min. The 29-mer RNA present in each lane was quantitated as a fraction of the total in each lane (U29-G37), normalized for the fraction remaining in the chase lane and plotted as a function of reaction time on a semilog plot to derive the rate of escape, kobs (•, noncomplementary bubble scaffold; {circ}, fully complementary scaffold).

 
The synthetic transcription bubble present in the nucleic acid scaffold introduces energetic barriers to transcription downstream of the site of reconstitution that become manifested as RNA products shorter than the expected 37-mer RNA (Fig. 1B, lane 5). These shorter products only arose when scaf-folds containing an artificial transcription bubble were used (Fig. 1B, compare lanes 5 and 6) and probably were caused by the inability of the upstream edge of the bubble to reanneal at these positions. Similar barriers to transcription have been observed on templates with synthetic bubbles created using noncomplementary nontemplate strands (18). Since we needed to measure the rate of the addition of only the first nucleotide, these barriers did not pose a problem for our experiments (see below), since their effects were manifest only after RNAP had added 5 nt to the RNA.

In Vitro Reconstituted his PTCs Were Kinetically Similar to PTCs Initiated at a Promoter—The reconstituted his PTCs exhibited kinetic properties similar to PTCs initiated from a promoter. Incubation of the rPTCs with GTP (10 µM) and ATP (100 µM) resulted in RNA extension (Fig. 1C). The amount of unextended RNA expressed as a fraction of the total in each lane after correcting for RNA not incorporated into ECs (Fig. 1C, lane C) was plotted as a function of time (Fig. 1C). A large fraction (~0.9) of the hairpin-containing PTCs elongated with a slow rate (kobs, 0.011 ± 0.003 s-1; t1/2, 63 ± 17 s), similar to prior observations for escape from the his pause (range of t1/2, 50–85 s) (6, 9). We confirmed this finding by measuring the pause half-lives of rPTCs and promoter-initiated PTCs in reaction mixtures containing both complexes together (t1/2 for both complexes = 72 ± 4 s; data not shown). Further, there was no significant difference in the kinetics of pause escape whether the PTCs were reconstituted on the partially noncomplementary (bubble) or fully complementary scaffolds (Fig. 1C).

PTCs Reconstituted Directly at the Pause Site or Upstream of the Pause Site Behaved Similarly—Because all previously studied PTCs were formed by synthesis of RNA preceding the pause site, we wanted to confirm that PTCs formed directly at the pause site in the absence of NTPs mimicked natural PTCs. To that end, we compared pause escape in ECs reconstituted at the pause site using RNA29 with ECs reconstituted with the 3' nt one or two positions before the pause site (RNA28 and RNA27, respectively) (Fig. 2), using a limited step transcription assay. The RNA27- and RNA28-containing complexes were assembled in the absence of NTPs and then advanced to the pause site at position U29 by incubating with UTP (RNA28) or with CTP and UTP (RNA27). Comparison of the kinetics of pause escape by these complexes revealed no significant differences in the rate of escape from the pause (Fig. 2; kobs, ~0.01 s-1), indicating that the behavior of these complexes at the pause is the same regardless of how they reached the pause site. We note that this may not be true of backtracked pause sites. This is important, because PTCs formed by direct reconstitution cannot contain PPi, whereas those formed upstream of the pause site could contain PPi, which conceivably could be important in PTC formation.

LSRA Revealed the Effect of the Pause Hairpin—Since disruption of the hairpin in promoter-initiated PTCs reduces pausing by a factor of ~10 (6), we wanted to test if the same is true for rPTCs that lack the RNA upstream of the hairpin ordinarily present in PTCs initiated from a promoter. To accomplish this, we adapted the limited-step transcription assay (5) to allow labeling of only active ECs (see "Experimental Procedures"), thereby allowing more accurate quantitation and measurement of both PTC formation and pause escape. We call this method LSRA. LSRA also allows ECs to be reconstituted upstream of the pause site, thus avoiding the complication of backtracking when PTCs lacking a hairpin are reconstituted directly at the pause. We could then use LSRA to test the contribution of the hairpin to pausing at the his pause site. A 27-mer RNA that forms the pause RNA hairpin with a 3'-end located 2 nt upstream of the pause site (RNA27, Fig. 3A) was labeled by incorporating [{alpha}-32P]CMP during reconstitution (Fig. 3B; LSRA step 1) and then elongated through the pause site in the presence of UTP and GTP to make the 31-mer RNA product (Fig. 3B; LSRA step 2). rPTCs formed in step 2 of LSRA escaped from the pause at a single rate (kobs, 0.01 ± 0.002 s-1; t1/2, 78 ± 8 s), similar to rPTCs formed by direct reconstitution (Fig. 1). This result rules out a transient contribution of PPi to pause lifetime, such as might occur if slow PPi release were rate-limiting for pause escape in the LSRA but absent in the direct reconstitution assay (Fig. 1).


Figure 2
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  		FIGURE 2.
Pause escape was indistinguishable whether PTCs are reconstituted directly at the pause or upstream of the pause. A, ECs were reconstituted 2 nt upstream of the pause site using the DNA scaffold (ntDNA, 5051; tDNA, 5420; RNA27, 4867) (Table 1) as in Fig. 1A, advanced to the pause site at U29 with CTP and UTP, and then elongated in the presence of 10 µM GTP. The 29-mer RNA present in each lane was quantitated as a fraction of the total in each lane (C28-G31; see "Experimental Procedures"), normalized for the fraction remaining in the chase lane, and plotted as a function of reaction time on a semilog plot to derive the rate of pause escape. B, ECs were reconstituted as in Fig. 2A, but 1 nt upstream of the pause site using RNA28 (Table 1), and advanced to the pause site using UTP, and the rate of pause escape was determined as in Fig. 2A. C, PTCs were reconstituted at the pause site as in Fig. 1A using RNA29 (Table 1), and the rate of pause escape was determined as in Fig. 2A.

 


Figure 3
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  		FIGURE 3.
Effect of the pause hairpin on rPTC transcription using LSRA. A, representation of the DNA scaffold (ntDNA, 5069; tDNA, 5420), hairpin RNA (RNA27, 4867), and no-hairpin RNA (RNA12, 4878; indicated on RNA27 by the gray area). Elongation of RNA27 or RNA12 by RNAP is shown in lowercase type. B, the experimental set up for LSRA is schematically depicted. ECs reconstituted with unlabeled RNA27 or RNA12 were advanced to positions C28 and C13, respectively, in the presence of [{alpha}-32P]CTP. These ECs were then elongated in the presence of GTP (10 µM) and UTP (1 mM) for short time intervals using a rapid quenched-flow apparatus. The reaction was quenched with 2 M HCl and neutralized with 3 M Tris; samples were processed as described under "Experimental Procedures"; and the RNA products were separated on 15% denaturing polyacrylamide gel. C, gels were quantitated as described under "Experimental Procedures." The amounts of pause hairpin RNA ({circ}, RNA29) and no-hairpin RNA (•, RNA14) were calculated as a fraction of the total in each lane and plotted versus reaction time. The disappearance of RNA14 was fit to a triple exponential and RNA29 to a single exponential. The rate of escape (kobs) and the fraction (f) for each species are indicated on the plot.

 
To determine the contribution of the hairpin, we measured the rate of pause escape using ECs containing a 12-mer RNA that lacked the pause hairpin (RNA 12, Fig. 3A). Based on previous results (5), we expected the no-hairpin RNA to generate PTCs but to escape ~10-fold faster than hairpin-containing PTCs. We also expected some fraction of the PTCs to bypass the pause site. Indeed, a significant fraction of the no-hairpin PTCs escaped from the pause site at a rapid rate that could only be measured using a quenched-flow apparatus (see "Experimental Procedures"). ECs containing the 12-mer RNA escaped from the pause site with at least three different rates (Fig. 3C), suggesting the presence of at least three kinetically distinguishable populations. The species that escaped with the slowest rate (kobs, 0.09 s-1) constituted 60% of the population and was still ~10-fold faster than the rate of escape from the pause site in the presence of the hairpin. Thus, the rPTCs exhibit a pause hairpin effect comparable with our previous estimate of the hairpin effect in promoter-initiated PTCs (4, 6). The two different rates observed for 40% of ECs that did not pause suggests the existence of two kinds of bypass ECs (see "Discussion").


Figure 4
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  		FIGURE 4.
rPTCs were resistant to pyrophosphorolysis and GreB-stimulated transcript cleavage. A, rPTCs are not substrate for pyrophosphorolysis. ECs reconstituted with RNA14 (3'-end in i + 1 site; top gel panel) or RNA29 (bottom gel panel) and DNA scaffold shown in Fig. 1A were incubated with PPi (1 mM) for the indicated times, and RNA products were separated on a 15% denaturing polyacrylamide gel. The 14-mer (•) and 29-mer ({circ}) RNA were quantitated as described under "Experimental Procedures," expressed as a fraction of the total in each lane, normalized to the amount at time 0, and plotted as a function of reaction time. *, the variation in amount of RNA29 in the 270 s lane is a consequence of loading error and not due to cleavage, since we did not see any cleavage products. B, rPTCs are resistant to GreB-mediated transcript cleavage. Backtracked ECs reconstituted containing a control 11-nt RNA with 2 bases at the 3'-end mismatched to the template strand (top gel panel, lanes 1–4) or RNA29 (bottom gel panel, lanes 1–4) and DNA scaffold as in Fig. 1A were incubated with GreB (4 µM) for the indicated times, and RNA products were separated on a 15% denaturing polyacrylamide gel. ECs were incubated with NTPs (GTP and UTP, 100 µM each; lane 5) or with GreB and NTPs (lane 6). The 11-mer (•) and 29-mer ({circ}) RNA were quantitated as a fraction of the total in each lane as described under "Experimental Procedures," normalized to the amount at time 0, and plotted as a function of reaction time. Lanes 5 and 6 in the top panel were moved to their present location from elsewhere on the same gel to align with the lanes in the lower panel.

 
The rPTCs Were Resistant to Pyrophosphorolysis and GreB-stimulated Transcript Cleavage—rPTCs exhibited several characteristics that were similar to promoter-initiated PTCs. For instance, promoter-initiated PTCs are not substrates for pyrophosphorolysis (7). The reconstituted his PTCs were insensitive to pyrophosphorolysis compared with no-hairpin PTCs, which were substrates for pyrophosphorolysis (Fig. 4A). Backtracked ECs are also not substrates for pyrophosphorolysis. To rule out the possibility that rPTCs were backtracked, they were subjected to GreB-mediated transcript cleavage. In a control experiment, ECs reconstituted in the backtracked state with the 3'-end of the RNA mismatched to the template strand were readily cleaved in the presence of GreB (Fig. 4B, top). As expected, ECs with backtracked control RNA were able to elongate only in the presence of GreB and added NTPs (Fig. 4B, compare lanes 5 and 6). The rPTCs were insensitive to the action of the cleavage factor GreB and were not dependent on GreB for nucleotide addition (Fig. 4B, bottom). Both of these results provide strong evidence that the active center of the rPTC is similar to that of a promoter-initiated PTC.


Figure 5
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  		FIGURE 5.
NusA enhanced pausing by rPTCs. A, rPTCs respond to NusA. rPTCs (~20 nM; as in Fig. 3) were elongated with GTP (10 µM) in the absence ({circ}) or presence (•) of NusA (100 nM). Aliquots were removed, and RNA was separated on a 15% denaturing polyacrylamide gel. The gel was quantitated as described under "Experimental Procedures," and the fraction RNA29 was plotted as a function of reaction time. A representative gel of an experiment done in triplicate is shown, and the half-life reported represents the average with S.D. B, ECs (~20 nM), initiated from the T7A1 promoter and halted at position A29 were elongated through the his pause site at position U71 with ATP, CTP, UTP (200 µM each), and GTP (10 µM) in the absence ({circ}) or presence (•) of NusA (100 nM). Samples were removed and processed, and RNA products were separated on an 8% denaturing polyacrylamide gel. The positions of the halted, paused, terminated and run-off (RO) RNA bands are indicated. The gel was quantitated as described under "Experimental Procedures," and the fraction of U71 pause RNA was plotted as a function of reaction time. A representative gel of an experiment done in triplicate is shown, and the half-life reported represents the average with S.D.

 
NusA Enhanced Pausing by rPTCs—The transcript elongation factor NusA increases pause dwell time at the his pause by a factor of 2–4-fold in a pause hairpin-dependent manner (4, 9). We therefore tested the ability of NusA to increase pause lifetime by rPTCs. ECs reconstituted 2 nt upstream of the pause using scaffolds as in Fig. 3 and elongated through the pause site exhibited an ~3-fold enhancement of pause lifetime by NusA (Fig. 5A). These results are similar to control experiments in which NusA enhanced pausing by promoter-initiated ECs by a factor of ~4 (Fig. 5B). These results unambiguously establish that upstream RNA is not required for NusA action at the his pause site. The slightly lesser NusA effect on the rPTC relative to a promoter-initiated PTC could reflect an ability of upstream RNA to enhance NusA action modestly, for instance by increasing NusA affinity for the PTC. However, in experiments using other DNA scaffolds but the same RNA (data not shown), we have observed no reproducible difference in NusA effect relative to promoter-initiated PTCs. Thus, we conclude that the ability of NusA to increase pause dwell time reflects direct interaction with the pause hairpin or RNAP (7), with little if any contribution of upstream RNA. These observations further validate the similarity of rPTCs to promoter-initiated PTCs.

Contribution of the Nucleic Acid Architecture to Pausing by rPTCs—Having established that rPTCs recapitulate the properties of naturally formed PTCs, we next used rPTCs to test the effects on pausing of the position of DNA strand separation, the length of downstream DNA, and potential RNA:DNA hybrid length. There is much interest in the position at which the downstream DNA separates in active and paused ECs as strand separation occurs from 0 to 3 bp downstream of the NTP-binding site in different EC models or crystal structures (19–22) and has been proposed to allow NTP pairing to DNA prior to translocation into the active site (23). Chemical probing suggests the his PTC is unpaired at +1 but not +2 (17), and the effects on pausing of sequence variants suggests that downstream DNA from +1 to ~+10 influences pausing, although this is not consistent with the ease of melting (3). We varied the potential location of the downstream edge of the transcription bubble by changing the sequence of the nontemplate DNA (Fig. 6A). Varying the site of potential pairing from -3 to +1 had no effect on pausing, whereas moving it forward to +2 and beyond decreased dwell time significantly. This suggests that DNA in the his PTC ordinarily is unpaired at +1, but not beyond +1, which is consistent with all studies of bacterial ECs (e.g. see Refs. 21 and 22), and of the his PTC in particular (17).

Interestingly, increasing the length of the downstream DNA past +15 decreased pause half-life significantly (Fig. 6B), and even 50 bp of downstream DNA did not recapitulate the half-life of control +15 rPTCs or promoter-initiated PTCs (t1/2, 74 ± 6 s in side-by-side controls at 10 µM GTP; this value is taken as 1 in Fig. 6). This suggests that contacts of RNAP to a downstream DNA end at +15 increased pause lifetime, either by inhibiting forward translocation or by favoring the active-site rearrangement at the pause, for instance by contacts to the clamp domain (see "Discussion").

The failure of the +50 bp downstream DNA to recapitulate natural PTC behavior caused us to wonder if restricting the hybrid length to 9 bp might affect pausing (although chemical probing suggests that the his PTC hybrid is ~9 bp) (17). Accordingly, we tested variant scaffolds that allowed potential hybrid lengths from 8 to 11 bp (Fig. 6C). Interestingly, reducing hybrid length to 8 bp did not affect pausing, with both 8- and 9-bp hybrids giving pause lifetimes close to natural his PTCs. This suggests that either an 8- or 9-bp hybrid can reconstitute into a pretranslocated EC (the natural register of the his PTC) if given a proper DNA scaffold. This contrasts with a recent suggestion that 8- or 9-bp hybrids form pretranslocated or posttranslocated ECs, respectively (24). However, allowing the hybrid to be 10 bp increased pause lifetime by a factor of ~3, regardless of exact sequences at -10. Allowing the hybrid to be 11 bp had no additional effect. These results suggest that hyperextension of the RNA:DNA hybrid to 10 bp may play a role in pausing (25, 26) (see "Discussion").


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The results from this study lead to four major conclusions. First, PTCs can be directly reconstituted at the pause site using nucleic acid scaffolds that contain the pause hairpin but no upstream RNA, and these rPTCs behave kinetically like PTCs initiated from a promoter. Second, PPi plays no role in determining pause lifetime. Third, NusA increases the lifetime of his rPTCs lacking upstream RNA to about the same extent at promoter-initiated PTCs containing a pause RNA. Fourth, the location of downstream DNA melting, the length of downstream DNA, and the potential length of RNA:DNA hybrid all can influence pause escape rates.


Figure 6
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  		FIGURE 6.
Contribution of nucleic acid scaffold architecture to pausing. Pause half-life was measured as in Fig. 3 and plotted relative to rPTCs using the DNA scaffold (ntDNA, 5069; tDNA, 4891; and RNA27, 4867; t1/2 = 74 ± 6 s). A, Opening of the downstream edge of the transcription bubble enhances pause escape. Pause half-life was measured in ECs reconstituted 2 nt upstream of the pause on scaffolds with different downstream edges in the artificial bubble relative to the pause 3'-nt at -1 (ntDNA: -3, 5071; -2, 5070; -1, 5069; +1, 5051; +2, 5056; +3, 5057; +4, 5058; tDNA: 5420; RNA 29; Table 1). Each scaffold contained 15 bp of DNA downstream of the pause site. The noncomplementary region of the nontemplate strand in the scaffolds is highlighted in gray. The RNA:DNA hybrid junction with the -10 base indicated (relative to the pause RNA 3'-nt) is enclosed by the dashed line. B, effect of downstream DNA length on pause half-life. ECs were reconstituted using DNA oligonucleotides with different lengths of DNA downstream of the pause site (DS; 15 bp tDNA/ntDNA, 4891/5069; DS 25 bp, 4897/5072; DS 35 bp, 4901/5073; DS 50 bp, 5040/5075; Table 1) and RNA that can form a 9-bp hybrid at the pause (RNA27, 4867). C, effect of RNA:DNA hybrid length on pause escape. ECs were reconstituted with scaffolds containing 15 bp of downstream DNA and RNA that can form an 8-bp (ntDNA/tDNA/RNA, 5051/4891/4870), 9-bp (5069/4891/4867), 9*-bp (5069/5420/5422), 10-bp (5069/5420/4867), 10*-bp (5069/4891/5422), or 11-bp (5051/5421/4867) RNA:DNA hybrid. The gray and open bars represent ECs assembled on scaffolds with 35 and 50 bp of downstream duplex DNA, respectively, and with a potential to form a 10-bp RNA:DNA hybrid. The sequence of the RNA:DNA hybrid junction (top, tDNA; bottom, RNA) with the position of the -10 base indicated by the dot is shown above each bar. *, RNA sequence variants different from wild type.

 
EC Reconstitution Can Be Used to Assemble rPTCs That Contain a Pause RNA Hairpin—The reconstituted his PTC exhibited several previously characterized properties of hairpin-stabilized PTCs initiated from a promoter. First, pause lifetime was increased by the pause RNA hairpin. Like PTCs initiated from a promoter, a vast majority of the rPTCs (~90%) escaped from the pause as a homogenous species with a single slow rate (kobs, ~0.01 s-1 at 10 µM GTP) ~8 times slower than ECs/PTCs reconstituted with an RNA that lacked the pause hairpin. At least three kinetically distinct species were observed using ECs/PTCs lacking the pause hairpin (Fig. 3B). These three species probably reflect reconstitution of RNAP into three different conformational states with respect to the location or orientation of the 3'-end of the transcript: posttranslocated, pretranslocated, and paused. The rapidly elongating species (kobs, >200 s-1) is likely to be posttranslocated and poised to bind NTP and undergo nucleotide addition. The species with a rate of ~1 s-1 probably represents the pretranslocated state that requires coupled translocation and NTP binding prior to nucleotide addition, which may be rate-limiting at this NTP concentration. If this is true, both fast species may represent states of the EC on the active nucleotide addition pathway that bypass the pause site without undergoing rearrangement into the paused state. The species with a slow rate (kobs, 0.085 s-1) would then correspond to the PTC. Elimination of the pause hairpin is known to increase pause escape but does not eliminate pausing (5). This slow species is ~8-fold faster than the hairpin-containing PTCs (kobs, 0.011 s-1; Fig. 1C), giving a lower estimate of the pause hairpin effect similar in magnitude (~10x) to that observed in previous experiments when pause hairpin formation is disrupted by substitutions (4) or by annealing of antisense oligonucleotides (6).

The hairpin-containing rPTCs were resistant to pyrophosphorolysis and to GreB-mediated transcript cleavage, indicating that the nature of the active site and location of the transcript 3'-end is in the same conformation as the his PTCs initiated from a promoter (6). Thus, the rPTCs bear significant similarities to their promoter-initiated counterparts in their kinetic properties and active site conformation, confirming that they are reconstituted in the paused conformation.

PPi Is Not Involved in Determining Pause Lifetime—Following NTP addition, a PPi release-mediated protein conformational change has been proposed to trigger translocation of the 3'-end of the transcript to allow the next cycle of nucleotide addition for T7 RNAP (27). This was based on the observation of posttranslocated ECs in crystals containing PPi and in pretranslocated state in the absence of PPi. If PPi release were rate-limiting for translocation of multisubunit RNAPs, then pause escape could be delayed, because translocation is inhibited until PPi is released. However, the fact that PTCs reconstituted directly in the absence of PPi or by LSRA show the same pause escape rates (Figs. 1, 2, 3) rules out involvement of PPi in determining pause lifetime. It remains possible that a delay in PPi release could provide time for PTC formation during active transcription, which would not be a factor in direct reconstitution. It also is possible that a block to translocation, especially to translocation stimulated by NTP binding (28–30), contributes to pausing, but this block cannot require a PPi-bound state.

RNA Upstream of the Pause Hairpin Is Not Required for Pause Stabilization or for NusA-mediated Enhancement of Pausing—The his rPTCs lack the RNA upstream from the pause hairpin that is present in promoter-initiated PTCs (Fig. 1A) (4, 6, 9) and that may potentially interact with RNAP or with NusA. RNAP contacts to upstream RNA (-30 to -45 in nonhairpin RNA) were postulated by Chamberlin and co-workers (31) based on RNA footprinting studies. NusA contacts to RNA upstream of the nut RNA hairpin were detected in photocross-linking experiments (32) and are expected based on the extended RNA binding surface of NusA (33). However, rPTCs lacking RNA upstream of the pause hairpin exhibited a pause hairpin effect similar to natural PTCs (Fig. 3) and an enhancement of pause half-life by NusA similar to natural PTCs (Fig. 5). Thus, the RNA upstream of the hairpin is dispensable for pausing or NusA-mediated pause half-life enhancement.

Scaffold Architecture Affects Pause Escape—Although we observed the same basic features of pausing, namely slow escape by a fraction of RNAPs encountering the pause site and inhibition of escape by the pause hairpin, on a variety of scaffolds, several aspects of scaffold architecture had significant effects on the rate of pause escape (Fig. 6). These include the position at which DNA strand separation occurs relative to the active site, the length of downstream DNA, and the potential length of the RNA:DNA hybrid.

Our results confirm prior evidence that the downstream DNA ordinarily is paired at +2 in the his PTC (17), because forcing strand opening further downstream accelerated pause escape (Fig. 6A). This is consistent with a proposal from Burton and co-workers (23) that collapse of the front edge of the transcription bubble may lead to pausing by blocking NTP pairing to downstream DNA prior to translocation into the active site. Such a view also could explain detection of strand separation at +2 or +3 in some ECs (17, 19). However, bubble collapse could instead affect a network of interactions involving the trigger loop, bridge helix, and clamp domain proposed to affect pausing (34). Additionally, this result could be explained by a shift in the energetics of translocation caused by downstream strand opening. The his PTC may be trapped in the pretranslocated register but could be driven into the posttranslocated register (and thus to pause escape) on an artificially unpaired scaffold by forward translocation that would reestablish an optimal transcription bubble and contacts of RNAP to the bubble junctions. Future studies of rPTCs may help distinguish these models in the context of a simple pause mechanism.

The effect of downstream DNA length (Fig. 6B) would also be consistent with an ability of forward translocation to promote pause escape if translocation is rate-limiting for pause escape. Downstream DNA contacts appear to extend out to at least +18 based on the RNAP footprint on DNA in the his PTC (5). When downstream DNA is shorter than 20 bp (e.g. the 15-bp downstream DNA in the minimal scaffold) (Figs. 3 and 6), RNAP forward translocation and pause escape may be disfavored, because front edge contacts would be lost. However, some of these contacts are made to the clamp domain, whose repositioning has been proposed to stabilize the his PTC (7). Thus, downstream DNA truncation also could increase pause lifetime by stabilizing a paused clamp position.

Finally, the effect of potential RNA:DNA pairing at -10 is intriguing (Fig. 6C). Since the his PTC is pretranslocated rather than backtracked (8), this suggests that a 10-bp RNA:DNA hybrid may be at least partially formed in the his PTC. Ample precedent exists for extension of the hybrid to lengths greater than 9 bp, but such extension appears to destabilize ECs due to structural rearrangement, such as movement of the lid element that ordinarily encloses the separated nascent RNA (26, 36). A 10-bp hybrid would reduce the spacer to 1 nt, which also would cause structural rearrangement in the RNA exit channel, because the hairpin or hybrid would clash with the lid. The pause-enhancing effects of pairing at -10 are consistent with a model in which rearrangements in the RNA exit channel caused by pause hairpin formation influence the active site via repositioning of the clamp domain (8).

The various effects on pause escape of front edge DNA strand separation, downstream DNA length, and potential RNA:DNA hybrid length beautifully illustrate the counter-balancing contributions of RNAP-nucleic acid interactions to the lifetime of the PTC that operate within the context of a simple pause mechanism. All are likely to be influenced by nucleic acid sequences at the sites of interaction and thus to allow a wide range of RNAP behavior to be fine tuned for a particular function and transcriptional unit by evolutionary pressure. Thus, the near identity of behavior we observed in a minimal rPTC (e.g. Fig. 1) versus natural PTCs probably reflects these tradeoffs (e.g. a short downstream DNA may compensate for the lack of potential hybrid pairing at -10). Exploiting these tradeoffs may aid design of scaffolds optimal for particular experimental goals, such as a minimal rPTC suitable for crystallographic study.

LSRA Is a Versatile Tool to Study Mechanisms of Transcript Elongation in Vitro—We originally developed a limited-step transcription assay as a method to study human RNAPII PTCs using promoter-initiated ECs and stepwise transcription (37), and it has been used since to study human RNAPII elongation kinetics (29). With the addition of EC labeling during reconstitution, the versatility of the method has been greatly increased, and LSRA now allows more accurate measurement of (i) the rate of isomerization into the his pause (e.g. at U29) by delaying the addition of the next NTP (e.g. GTP), (ii) the rate of escape from the pause, and (iii) the rate of bypass of the pause. In addition, it should now be possible to incorporate biophysical probes of RNAP and nucleic acid dynamics (e.g. fluorescent probes) into kinetic experiments on multisubunit RNAPs. Since gene expression is controlled in part during transcript elongation in both prokaryotes and eukaryotes, understanding how transcript elongation is regulated is of great importance. Transcriptional pausing (hairpin-stabilized, backtracked, or factor-stabilized) is an underlying mechanism that allows regulation of transcript elongation. The mechanistic approach described here should allow dissection of the effects of regulatory proteins and factors on transcript elongation and provide a better understanding of the control of gene expression.


   FOOTNOTES
 
* This work is supported by National Institutes of Health Grant GM38660 (to R. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported in part by NIGMS, National Institutes of Health, National Research Service Award T32 GM07215. Present address: Epic Systems, 1979 Milky Way, Verona, WI 53593. Back

2 To whom correspondence may be addressed: Dept. of Biochemistry, 420 Henry Mall, University of Wisconsin-Madison, Madison, WI 53706. Tel.: 608-265-8475; Fax: 608-262-9865; E-mail: landick{at}bact.wisc.edu. 3 To whom correspondence may be addressed: Dept. of Biochemistry, 420 Henry Mall, University of Wisconsin, Madison, WI 53706. Tel.: 608-265-8709; Fax: 608-262-9865; E-mail: palangat{at}facstaff.wisc.edu.

4 The abbreviations used are: RNAP, RNA polymerase; LSRA, limited-step reconstitution assay; EC, elongation complex; PTC, paused transcription complex; rPTC, reconstituted paused transcription complex; tDNA, template DNA; ntDNA, nontemplate DNA; nt, nucleotide(s); RB, reconstitution buffer. Back

5 K. S. Ha and R. Landick, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank members of the Landick laboratory for critical reading of the manuscript, helpful suggestions, and many productive discussions.



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