Nuclease cleavage of the upstream half of the nontemplate strand DNA in an Escherichia coli transcription elongation complex causes upstream translocation and transcriptional arrest.

We tested the susceptibility of nucleic acid strands in a halted transcription elongation complex to digestion by micrococcal nuclease (MN). The 16-nucleotide nascent RNA was protected within RNA polymerase. A 27-28-nucleotide template strand DNA fragment also was resistant to MN digestion. However, the upstream half of the nontemplate DNA within this region was digested rapidly by MN, suggesting that the nontemplate strand emerges from the RNA polymerase near the middle of the melted transcription bubble with the bases oriented away from the enzyme surface. MN cleavage of the exposed nontemplate DNA shifted polymerase backward, making it unable to extend the RNA chain. However, the MN-trimmed G16 complexes could be reactivated by GreB-stimulated cleavage of the nascent RNA. These results favor a model of transcriptional arrest involving upstream slippage of RNA polymerase along the RNA and DNA chains. They also suggest that the exposed segment of nontemplate DNA may directly or indirectly stabilize the lateral position of the transcription complex along the DNA.

Transcribing RNA polymerase contacts ϳ30 bp 1 of DNA, although the length of DNA protected from cleavage agents can vary by as much as ϳ10 bp in transcription complexes halted near initiation, pause, termination, or arrest sites (1)(2)(3)(4)(5)(6)(7)(8)(9). An ϳ18-bp segment of DNA from near the upstream edge to the middle of this footprint is melted to form the transcription bubble, as indicated by both topological and chemical reactivity assays (7, 9 -12). The reactive region also can vary by Ϯ5 bp in some halted complexes (7,9,11), although this has yet to be tested by topological measurement. To maintain the transcription bubble, the nontemplate DNA strand must be separated from the template strand as polymerase moves forward and then reanneal after the template strand is transcribed. Previous findings led us to suggest that, whereas strand separation occurs within polymerase, ϳ10 -15 bp upstream from its forward edge on DNA, the strands reanneal passively as the template DNA exits upstream (Refs. 11 and 13; see Fig. 1).
This hypothesis predicts that a part of the nontemplate DNA may be exposed on the outside of the transcription complex, rather than protected within it. The chemical reactivity of the nontemplate DNA is consistent with this view. Within the transcription bubble, bases on the nontemplate strand are highly reactive with single strand-specific reagents such as KMnO 4 , dimethylsulfate, or diethylpyrocarbonate (7,9,11,12), whereas the phosphodiester backbone is mostly protected from cleavage by HO⅐ (hydroxyl radical) (3,7). The template DNA in contact with RNA polymerase also is protected from HO⅐ generated in solution; however, it is readily cleaved by HO⅐ generated from the intercalating reagent 1,10-phenanthroline copper, whereas the corresponding nontemplate DNA is not (14 -16). This suggests that interactions of the template strand bases with polymerase or with RNA mediate high-affinity binding of 1,10-phenanthroline copper, but that nontemplate strand bases either are loosely associated with polymerase or held in a channel accessible to small reagents such as KMnO 4 but not to the larger 1,10-phenanthroline copper. Although these findings are consistent with the idea that part of the nontemplate strand lies on the surface of RNA polymerase, they fall short of establishing it unambiguously or delineating which part is exposed. Furthermore, although the major sources of transcription complex stability recently were localized to contacts to the nascent RNA, the template DNA strand near the active site, and ϳ10 bp of duplex DNA in front of the transcription bubble (17), the possible role of the nontemplate DNA in stabilizing the position of polymerase along the DNA is unknown.
To address these questions, we examined the susceptibility of the nontemplate DNA to cleavage by micrococcal nuclease (MN). MN cleaves both RNA and DNA endonucleolytically by Ca 2ϩ -mediated attack of OH Ϫ at the 5Ј-position of the phosphodiester bond, yielding 3Ј-mononucleotides or -oligonucleotides (18 -20). Bases are bound without sequence specificity within a large (ϳ10 Å wide) hydrophobic pocket, whereas the scissile and 3Ј-phosphates make hydrogen bond contacts to MN near the rim of the pocket. These properties make MN an ideal probe for exposure of bases in the transcription complex.
We found that rapid MN cleavage of nontemplate DNA within the transcription bubble causes RNA polymerase to slide upstream along the DNA and RNA chains into an arrested state. An arrested RNA polymerase remains bound to the DNA and nascent transcript but is unable to extend the RNA chain because its 3Ј-OH has relocated outside of the active site (4,8,(21)(22)(23)(24)(25)(26)(27). Either active site slippage relative to the RNA or RNA slippage relative to the active site generates complexes in which the antiarrest factors GreA or GreB can stimulate internal cleavage of the RNA chain (28), thus restoring the ability of polymerase to extend it. We consider the implications of our findings for the mechanism of arrest under "Discussion."

MATERIALS AND METHODS
Reagents and Enzymes-ApU was purchased from Sigma; polynucleotide kinase from New England Biolabs; micrococcal nuclease from Boehringer Mannheim; purified NTPs from Pharmacia Biotech Inc.; and [␣-32 P]GTP and [␥-32 P]ATP from Amersham Corp. RNA polymerase was purified by the method of Burgess and Jendrisak (29) with the modifications described previously (28). GreA and GreB were purified after overproduction from plasmids pDNL279 and pGF103 as described previously (6,28).
Preparation of DNA Templates-DNA templates for transcription reactions were synthesized by polymerase chain reaction amplification from plasmid pCL185 (28) with primers that hybridized upstream and downstream from the T7A1 promoter-leader segment: primer 947, 5Ј-GAGAGACAACTTAAAGAGAC (91 bp upstream from the transcription start site); and primer 1498, 5Ј-GGTGTTTAAATTTGAACGC (50 bp downstream). The polymerase chain reaction product is 142 bp long and yields a 50-nt-long runoff transcript (see Fig. 1).
The G16 complex was purified and exchanged to MN buffer by passing through a 500-l Sepharose G-50 spin column equilibrated with MN buffer (10 mM Tris-HCl, pH 7.5, 20 mM ammonium acetate, 0.2 mM EDTA, 2 mM ␤-mercaptoethanol, 20 g of acetylated bovine serum albumin/ml, 2% (v/v) glycerol. After exchanging to MN buffer, CaCl 2 was added to G16 complex to a final concentration of 1 mM. G16 complexes were then digested with 10 units of MN/ml (final) at 37°C for the time desired. Digestions were stopped by adding 3 mM EGTA (final) to the mixture.
Agarose Gel Electrophoresis of Transcription Complexes-G16 complexes before and after MN digestion were electrophoresed through a 50 ϫ 75-mm 4% NuSieve agarose gel (FMC) cast on the hydrophilic side of Gelbond film (FMC; ϳ12 ml of 4% agarose in 0.5 ϫ TBE (1 ϫ TBE is 89 mM Tris borate, pH 8.3, 2.5 mM EDTA)). Surface tension suffices to adhere the gel to the plastic. Samples were prepared by addition of glycerol to 10%, heparin to 100 g/ml, and xylene cyanol and bromphenol blue to 0.05% each. Electrophoresis was performed with the gel submerged in 0.5 ϫ TBE buffer at room temperature and 5 V/cm. After migration of the bromphenol blue to the bottom of the gel (75 mm), the gel was soaked in 0.5 g of ethidium bromide/ml in water to stain DNA. After photography, the gel was soaked in 0.5% Coomassie Brilliant Blue R (Sigma) in 50% methanol, 10% acetic acid for 60 min and destained by gentle shaking overnight submerged in a covered tray of 20% methanol, 10% acetic acid, also containing a small piece of open cell polyurethane foam. Adherence to Gelbond film is essential to prevent disintegration of the agarose gel, which becomes brittle in the stain and destain solutions.
Recovery, End Labeling, and Sequencing of the MN-digested DNA-To recover the MN-digested DNA, the digested G16 complexes were extracted once with phenol-chloroform, the aqueous layer was then extracted once with chloroform, and the DNA was recovered by ethanol precipitation.
To end label the recovered DNA products, 1 pmol of MN-digested DNA (MN digestion generates 5Ј-OH) was incubated with 15 units of polynucleotide kinase (New England Biolabs) in buffer provided by the manufacturer and 2 l of [␥-32 P]ATP in a final volume of 15 l at 37°C for 30 min. The end-labeled DNA was purified away from the free [␥-32 P]ATP by centrifugation through a pre-equilibrated 150-l Sepharose G-10 spin column (Pharmacia) at 500 ϫ g for 5 min. The endlabeled DNA then was mixed with an equal volume of loading buffer (2 ϫ TBE, 50% (w/v) urea, 0.25% (w/v) each xylene cyanol and bromphenol blue), heated at 95°C for 5 min, and resolved on a 20% polyacrylamide (19:1 acrylamide:bisacrylamide), 7 M urea, 0.5 ϫ TBE gel. The gel was then exposed to Kodak XAR-5 film to locate the labeled single strand DNA. The radioactive bands were then excised as small gel slices and soaked in 1% SDS, 0.5 M ammonium acetate overnight at 37°C for elution. The supernatants were separated from the gel slices, extracted with phenol-chloroform, and then extracted to dryness with 1-butanol. The pellets containing the labeled, single strand DNAs were washed once with 70% ethanol, dried under vacuum, and resuspended in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA).
Chemical sequencing of purified 5Ј-end-labeled single strand DNAs was performed as described by Maxam and Gilbert (32). The sequencing products were then resolved on a 20% polyacrylamide sequencing gel (19:1 acrylamide:bisacrylamide) and visualized by exposing on Kodak XAR-5 films.
Elongation of MN-digested G16 Complex-After MN digestion, MgCl 2 was added to the trimmed G16 complex to a final concentration of 7 mM. Then the trimmed G16 complex was elongated in the presence of 600 M all four NTPs for 1, 5, and 30 min at 37°C with or without 0.12 M GreB.  (11,13). The boundaries of the G16 complex as measured by ExoIII footprinting are indicated by arrows. A summary of the reactivities of G16 (13) and 12 other halted elongation complexes (11) to KMnO 4 and diethylpyrocarbonate, reagents that are specific for unstacked bases, is plotted above and below the strands (arbitrary scale with 1 equal to strong reactivity). The protected region of template DNA probably reflects an ϳ8 bp RNA-DNA hybrid (11), although protection by strong interaction with RNA polymerase has not been ruled out.

RESULTS
The G16 Complex Is Stable after MN Digestion-To test whether the G16 complex is stable after MN digestion, we performed a digestion time course (Fig. 2). After successive intervals, an aliquot of the reaction was removed and stopped by addition of EGTA to 3 mM. We then electrophoresed the samples on a 4% agarose gel (see "Materials and Methods"). After electrophoresis, we stained the gel successively with ethidium bromide to detect the DNA ( Fig. 2A) and with Coomassie Brilliant Blue to detect the protein (Fig. 2B). Greater than 90% of the DNA template was incorporated into G16 complex ( Fig. 2A, lane 1). A small amount of the DNA was associated with a slower moving complex, probably the result of loading a second polymerase onto the promoter once the G16 complex formed (33). The second polymerase was released from the DNA by 2 min of MN digestion, since only a single MNtrimmed complex was evident at that point (Fig. 2, A and B,  lane 3). MN digestion was complete after 8 min when all the G16 complex was converted to a faster moving band and most of the ethidium bromide-stainable DNA was lost (Fig. 2, A and B, compare lanes 5 and 1).
In contrast, the 16-mer nascent RNA in G16 complexes was completely resistant to digestion by MN (Fig. 3A, lanes 2-7) but not after it was artificially dissociated from the complexes and gel-purified (Fig. 3B, lanes 2-9). Presumably the 16-nt nascent RNA remained within RNA polymerase and was protected from MN attack. Longer nascent RNAs also were cleaved by MN and shortened to a 20 -24-nt 3Ј-protected fragment (data not shown). However, the G16 nascent RNA no longer could be extended even after 2 min of MN digestion (Fig. 3A, lanes  8 -13), which was well before the DNA digestion was complete (ϳ8 min, see above). We conclude that MN digestion trims excess DNA from transcription complexes and efficiently converts them to arrested complexes.
To test this interpretation, we treated the G16-arrested complexes with the transcript cleavage factor GreB (Fig. 3C). At low molar ratio (Fig. 3C, lanes 2 and 3), GreB principally cleaved off a 6-nt fragment (Fig. 3C, b), leaving a 10-nt nascent RNA (Fig. 3C, a). A weaker cleavage released a 5-nt fragment, producing an 11-nt nascent RNA. At a higher concentration (Fig. 3C, lanes 4 and 5), GreB shortened these further to a 7-nt nascent RNA (Fig. 3C, c) with release of dinucleotides or trinucleotides (Fig. 3C, d). When GreB treatment was conducted in the presence of NTPs, the same initial cleavage product was evident, but the nascent RNA was rapidly elongated to an ϳ24-nt runoff RNA (Fig. 3C, lanes 6 -8). Treatment of MN-trimmed G16 complexes with GreA yielded similar results, although large 3Ј-cleavage products were not detected (data not shown). We conclude that G16 complexes were inactivated, but not dissociated, by MN digestion.
The inactivated complexes had features typical of arrested transcription complexes; they could resume elongation only   FIG. 3. Fate of nascent RNA in the MN-trimmed G16 complex. A, G16 RNA during and after MN treatment. The RNA was labeled with [␣-32 P]GTP during formation of the G16 complexes (see "Materials and Methods") and then exposed to MN in the same samples used to gen-erate the results shown in Fig. 2 (lanes 2-7). After addition of EGTA to stop MN, a portion of the samples was incubated with all four NTPs at 400 M (lanes 8 -14). B, digestion of free G16 RNA with MN. RNA was isolated by phenol extraction of G16 complexes and ethanol precipitation. The isolated G16 RNA was incubated for the times indicated without MN (lane 1) or with MN at 10 units/ml (lanes 2-5) or 100 units/ml (lanes 6 -9). C, treatment of MN-trimmed complexes with GreB. Complexes were incubated with increasing concentrations of GreB for 30 min at 37°C in the absence (lanes 2-5) or presence (lanes 6 -8) of 150 M NTPs. The assignments of bands to individual RNA species was made by comparison with GreA-generated cleavage products (not shown), the sequences of which were reported previously (28). The presence of one terminal phosphate on the 3Ј-proximal released RNA products causes their greater mobility relative to the 5Ј-proximal nascent RNAs, which contain OH groups at both ends (11, 28).
after GreB-induced nascent RNA cleavage, which produced large 3Ј-RNA fragments indicative of relocation of the active site over an internal phosphodiester bond of the nascent RNA. The 10-and 6-nt fragments produced suggest that the active site was positioned over the G10-A11 phosphodiester bond after MN treatment. Interestingly, prolonged incubation at 4°C of G16 complexes that have not been treated with MN also leads to arrest and slow, spontaneous cleavage between A11 and C12 on shifting to 37°C (28). The correlation of large fragment cleavage, arrest, and dwell time was discovered subsequent to publication (data not shown) and has been described by several other groups (5,8,21,(23)(24)(25)36). The 1-nt difference in cleavage position suggests that MN treatment causes RNA polymerase to slide backward further than occurs spontaneously.
The Upstream Half of the Nontemplate Strand in the Transcription Bubble of G16 Complex Is Cleaved by MN-In addition to differences in elongation potential and GreB cleavage, MN-trimmed complexes elongated only to position ϩ24, 6 nt short of the boundary of G16 complexes previously defined with ExoIII (Ref. 1; see Fig. 1). To determine whether this reflected differences in the protected DNA strands that remained in the MN-trimmed elongation complex, we analyzed their sequences. After extraction and recovery, the fragments were 5Ј-end-labeled and resolved on a 20% denaturing gel (see "Materials and Methods"). We observed nine bands ranging from ϳ12 to ϳ28 nt in size (Fig. 4A, bands a-i). We recovered all the radioactive bands from the gel and subjected them to chemical sequencing (see "Materials and Methods"). Comparison of these sequences with that of the 142-bp DNA template revealed that bands a-e and g were fragments protected from MN digestion by the G16 transcription complex (Fig. 4B). Minor bands f, h, and i yielded degenerate sequences that could not be matched to DNA within the G16 complex footprint (data not shown).
Comparison of the sequences of bands a-e and g with that of DNA within the G16 complex led to four main observations (Fig. 4B). First, bands a-c corresponded to the template strand DNA, and bands d, e, and g corresponded to the nontemplate strand. Second, all the bands shared the same downstream edge (Fig. 4B), indicating that the downstream DNA in the MN-trimmed G16 complex is tightly and uniquely associated with RNA polymerase. Third, the upstream half of the nontemplate DNA in the G16 complex was much more accessible to MN digestion than was the template strand DNA. Cleavage sites in the nontemplate strand DNA were within the transcription bubble (compare Figs. 1 and 4B), suggesting that the upstream half of the nontemplate strand in the bubble is exposed. Fourth, the upstream half of the nontemplate DNA in the transcription bubble could not be recovered from the MNtrimmed G16 complex. It is likely that this part of the DNA was released from the polymerase after MN cleavage and reduced to mononucleotides and dinucleotides that were lost during EtOH precipitation (see "Materials and Methods"). We conclude that the upstream portion of the nontemplate DNA strand is unnecessary for transcription complex stability, in agreement with the findings of Nudler et al. (17) that only downstream duplex DNA, the nascent RNA, and a short segment of the template DNA near the active site are required for stability of the transcription complex.
MN Digestion Caused Upstream Slippage of the G16 Complex on DNA and Transcriptional Arrest-A comparison of the G16 complex footprint determined previously with ExoIII ( Fig.  1) with that determined with MN (Fig. 4B) revealed an upstream shift of the contact of the polymerase to DNA by 5 or 6 bp with MN treatment. Since MN prefers single strand DNA to double strand DNA (20), we reasoned that the exposed upstream half of the nontemplate strand DNA in the transcription bubble was quickly cleaved by MN. After losing part of the transcription bubble, the G16 complex collapsed and slid backward on DNA (Fig. 5). This interpretation is consistent with backward movement of the active site by 6 nt on the RNA transcript and with formation of the arrested complex before the flanking DNA is completely digested around RNA polymerase (compare Figs. 2 and 3). It also explains why the upstream end of the dominant protected species on template DNA (Fig. 4, A and B, a) is located ϳ5 nt upstream from the ExoIII footprint boundary. Polymerase must shift upstream fast enough to protect these 5 nt. Likewise, the dominant nontemplate species (Fig. 3, A and B, e) extends 5 nt further upstream than band g, which could also reflect protection upon rapid upstream movement of the complexes. DISCUSSION We report here that the upstream half of the nontemplate DNA in the transcription bubble of a G16 halted elongation complex is exposed to attack by MN. The RNA transcript, the template strand DNA, and the other half of the nontemplate strand DNA, however, are relatively protected. MN digestion of FIG. 4. Chemical sequencing of MN-trimmed G16 DNA. A, radioactive labeling of MN-trimmed G16 DNA. The MN-trimmed G16 DNA was end-labeled with 32 P as described under "Materials and Methods" and resolved on 20% denaturing polyacrylamide gel. Bands d, e and g probably were less efficiently labeled compared with bands a-c because of the 3Ј-overhang of the template strand DNA (Fig. 3, A and B). The efficiency of bands d, e, and g labeling also may have varied because their 5Ј-ends differ. Bands a-c have the same blunt-ended 5Ј-OH. Thus, their relative intensity probably reflects the abundance of each individual band. B, representation of the DNA template used in the experiments. DNA sequences from -12 to ϩ30 relative to the transcription start site are shown. The lines above or below that DNA sequence indicate the fragments remaining after MN treatment of G16 complexes. The thickness of the lines represents the relative intensity of the bands. the exposed part of the nontemplate strand caused the G16 complex to collapse backward by ϳ6 bp into an arrested state. A 6-nt segment of nascent RNA also appeared to be reversethreaded through the active site during the collapse, since it was removed on incubation with the GreA or GreB transcript cleavage factors.
The conclusion that the entire RNA polymerase molecule moves backward relies on an assumption that ExoIII and MN would yield similar footprints if the complex did not move. We favor this interpretation, because both edges of the MN footprint are offset upstream by ϳ6 bp, so that both the ExoIII and MN footprints are ϳ28 bp (Fig. 4). It seems likely that this change reflects polymerase movement, rather than differential nuclease specificity, because ExoIII, which is double strandspecific and thus would stop when it encounters the edge of the single-stranded bubble, digests to ϩ1 of the template strand, whereas MN, which should attack exposed single strand DNA rapidly, predominantly cleaves the template strand DNA to only Ϫ5.
We will discuss the implications of these findings for three topics: the structure and stability of the transcription complex, the mechanism of transcriptional arrest, and the potential for the nontemplate DNA strand to be targeted by regulators of RNA chain elongation.
Transcription Complex Structure and Stability-Consistent with previous ideas (11,13), our results suggest that the up-stream portion of the nontemplate strand lies on the outside of RNA polymerase and reanneals passively with the template strand as the latter emerges from the transcription complex (Fig. 5). This view also is supported by the weak reactivity of the upstream part of the nontemplate DNA with HO⅐ within the generally protected footprint (7). It seems likely that the phosphate backbone of the nontemplate strand is most closely associated with RNA polymerase, since the bases throughout the OH-protected region are reactive to chemical probes such as KMnO 4 , dimethylsulfate, and diethylpyrocarbonate (7,9,(11)(12)(13). Most likely, the phosphate backbone anchors the downstream portion of the melted nontemplate strand in a channel large enough to let small molecules attack the bases but that is not accessible to MN (Fig. 5). Rapid attack of the unprotected nontemplate strand by MN, which binds a base within a hydrophobic pocket, suggests that the bases in this segment are directly exposed to solvent, perhaps with the phosphate backbone interacting with positively charged amino acid side chains on the surface of polymerase.
What contribution does the nontemplate strand make to stability of the transcription complex? Nudler and co-workers (17) recently established that elongation complexes require contact to only the downstream DNA duplex and singlestranded template from -1 to ϩ1 to retain function and saltstable binding. Hence the nontemplate strand cannot be essential to prevent dissociation of the complex. However, our results suggest it may be critical for the lateral stability of the transcription complex along the RNA and DNA chains. Since melting of a base pair at one end of the transcription bubble is accompanied by reannealing of another at the other end, the transcription complex may be able to occupy multiple positions along the RNA chain, one of which is competent for elongation and others of which would locate the active site over an internal phosphodiester bond in the RNA. Based on the ability of both transcript cleavage and pyrophosphorolysis to drive reverse translocation of RNA polymerase, Feng et al. (28) suggested that the distribution among these possible states would be determined by their relative stabilities and the kinetic barriers to interconversion, with the active state normally dominant. The slipped states would arise depending on sequences of the chains and dwell time at a particular RNA 3Ј-end. This idea is consistent with the observation that RNA polymerase sometimes enters unactivated states in kinetic analyses of chain elongation (34) and misincorporation (35). In this view, rapid reverse slipping of polymerase upon removal of the exposed nontemplate DNA strand by MN is most easily explained because the remaining nontemplate strand can re-pair with the template strand and increase the thermodynamic stability of the slipped state relative to the original G16 position (Fig. 5).
Mechanism of Transcriptional Arrest-A current model for transcriptional arrest is that relocation of the active site over an internal phosphodiester bond in the nascent RNA arises when part or all of the RNA polymerase slips backward along the DNA template, blocking subsequent nucleotide addition until transcript cleavage occurs (4, 8, 21-24, 26, 27). Reports that the RNA cleavage interval increases on arrest (5,8,21,(23)(24)(25) and that cleavage causes upstream shifting of the footprint of RNA polymerase on DNA in a nonarrested complex (13), but not in an arrested transcription complex (22), all favor this model. Here we find that an upstream shift in both edges of the DNA footprint of the polymerase is associated with arrest. Our results thus strongly support the idea that backward translocation of RNA polymerase along DNA and RNA chains can trigger transcriptional arrest. In this case slippage and arrest are caused by nontemplate strand removal, but similar backward slippage on transcriptional arrest also has Shaded oval, RNA polymerase. White channels, RNA and DNA binding grooves. In the G16 complex, the downstream DNA binding site covers 10 -13 bp of double strand DNA (see Fig. 1). Preferential cleavage in the upstream half of the single strand nontemplate DNA causes G16 complexes to collapse backward, leading to transcriptional arrest. The length of nontemplate strand DNA is heterogeneous, because initial MN cleavage at different sites becomes fixed after polymerase shifts backward and protects the nontemplate DNA against further digestion. been observed without MN treatment for both E. coli RNA polymerase (36) and mammalian RNA polymerase II (37).
A key question now is how these slippage events are related to the apparent discontinuous changes in the RNA and DNA footprints of RNA polymerase (so-called "inchworming") that have been found to be associated with pausing, arrest, and termination (5,6,8). Flexibility in RNA polymerase on encountering certain DNA or RNA sequences could lead to dislocation of the RNA 3Ј-end from the active site of the polymerase and subsequent sliding of the enzyme. Alternatively, sliding of RNA polymerase along the RNA and DNA chains when complexes are halted for study near regulatory sites may generate the inchworm footprints (36,37), reflecting a change in the energetic state of the transcription complex that foreshadows pausing, arrest, or termination.
Regulatory Factor Interaction with the Nontemplate Strand-The upstream half of the nontemplate strand recently has been found to be the target of a noncanonical activity of the E. coli 70 initiation factor (38): induction of pausing at position 16 of the late transcription unit (39). At least in these complexes, the nontemplate strand is exposed in a way that allows sequence-specific recognition by 70 . Thus, as in our finding that MN can digest this portion of nontemplate DNA (which appears to require outward orientation of the bases), 70 -induced pausing suggests that nontemplate bases are exposed to potential interactions with regulatory factors. These findings raise the speculative possibility that some factors that regulate transcript elongation could target the nontemplate DNA strand, inhibiting or promoting either forward or reverse translocation of the enzyme, and thus favoring pausing, arrest, termination, or rapid transcription, by influencing availability of the nontemplate DNA strand for re-pairing with the template DNA strand upon translocation.