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Bacterial RNA polymerase (RNAP) holoenzyme consists of a catalyticcore enzyme (α2ββ′ω) in complex with a σ factor. σ factors confer onthe core enzyme the ability to initiate transcription at specific promot-ers1. In Escherichia coli the primary σ factor is the σ70 subunit, whichmakes direct contact with the conserved –10 and –35 promoter elements1. During the transition from initiation to elongation, upon synthesis of a 9–11 nucleotide (nt) RNA transcript, the stability ofinteractions between σ70 and the remainder of the transcription com-plex decreases2–7, owing at least in part to displacement of a segmentof σ70 from the RNA exit channel by the nascent RNA8–11.Nevertheless, there is no obligatory mechanistic requirement forrelease of σ70 upon transition to elongation, and, in at least some tran-scription elongation complexes, σ70 remains associated with, andtranslocates with, RNAP12–16. A key question is whether retention ofσ70 confers distinct functional properties on transcription elongationcomplexes—that is, whether σ70 has functional roles in transcriptionelongation.

The strongest evidence indicating that σ70 can play a functional roleduring elongation comes from studies of the bacteriophage λ PR′ pro-moter. In particular, the expression of phage late genes from λPR′depends on a σ70-dependent pause that occurs during early elonga-tion, after RNAP has escaped from the promoter and synthesized a 16- or 17-nt transcript17. This pause is mediated by protein-DNAinteraction between σ70 and a DNA sequence element in the initiallytranscribed region that resembles a promoter –10 element14,17.Furthermore, the same module of σ70 that mediates interaction withthe –10 element of promoters mediates interaction with the pause-inducing sequence element14,17. Formation of the paused elongationcomplex at λPR′ is required for the phage λ Q antiterminator protein

(λQ) to gain access to and subsequently join the transcription com-plex, enabling RNAP to read through downstream terminators andtranscribe the phage’s late genes17. Similar regulatory pause-inducingsequence elements are present at the late promoters of other lambdoidphages, including phage 82, phage 80 and phage 21 (ref. 14).

The events that occur at the lambdoid phage late promoters raise thepossibility that other promoters bear DNA sequence elements thatinduce σ70-dependent pauses in early elongation. To investigate thispossibility, we examined transcription initiating from plac, a well-characterized bacterial promoter. Like λPR′, plac bears a DNAsequence element in the initially transcribed region that resembles apromoter –10 element. We provide evidence that this sequence ele-ment mediates a σ70-dependent pause in early elongation at plac, bothin vitro and in vivo. In addition, we demonstrate a functional correla-tion between the paused elongation complexes at plac and λPR′.Finally, we show, using biophysical analysis, that the pause-inducingsequence element at plac stabilizes the interaction between σ70 and theremainder of the elongation complex.

RESULTSA –10-like element mediates a pause at placWe noticed that the DNA sequence in the initially transcribed regionof plac resembles a promoter –10 element. In particular, the sequencefrom position +1 to +6 of plac, like the corresponding sequence ofλPR′, is a 3-of-6 match to the consensus –10 element and includes thetwo most highly conserved base pairs: an A at the second position anda T at the sixth position (Fig. 1a). There is also an extended –10 (TG)motif18 one base pair upstream of the sequence (at positions –3 and–2). We therefore wished to examine whether or not the –10-like DNA

1Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts 02115, USA. 2Howard HughesMedical Institute, Waksman Institute, Department of Chemistry, Rutgers University, Piscataway, New Jersey 08854, USA. Correspondence should be addressed toA.H. (ahochschild@hms.harvard.edu).

Published online 2 May 2004; doi:10.1038/nsmb757

The σ70 subunit of RNA polymerase mediates a promoter-proximal pause at the lac promoterBryce E Nickels1, Jayanta Mukhopadhyay2, Sean J Garrity1, Richard H Ebright2 & Ann Hochschild1

The �70 subunit of RNA polymerase plays an essential role in transcription initiation. In addition, �70 has a critical regulatoryrole during transcription elongation at the bacteriophage � late promoter, �PR′. At this promoter, �70 mediates a pause in earlyelongation through contact with a DNA sequence element in the initially transcribed region that resembles a promoter –10element. Here we provide evidence that �70 also mediates a pause in early elongation at the lac promoter (plac). Like that at�PR′, the pause at plac is facilitated by a sequence element in the initially transcribed region that resembles a promoter –10element. Using biophysical analysis, we demonstrate that the pause-inducing sequence element at plac stabilizes the interactionbetween �70 and the remainder of the transcription elongation complex. Bioinformatic analysis suggests that promoter-proximal�70-dependent pauses may play a role in the regulation of many bacterial promoters.

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element in the initially transcribed region of plac could mediate a σ70-dependent pause in early elongation.

As an initial test of this idea, we conducted single-round in vitrotranscription assays using either a λPR′ template or a constitutive ver-sion of plac, placUV5, which differs from wild-type plac by the substi-tution of two base pairs in the –10 element: G(–9)A and T(–8)A(ref. 19). To detect transcription pauses we carried out each assay as atime course, monitoring the RNA content of each reaction at discretetimes after the initiation of transcription (Fig. 1b). When the λPR′ tem-plate was used, RNA species of 16 and 17 nt in length appeared early inthe time course and disappeared over time, consistent with previousobservations20–23. When the placUV5 template was used, prominentRNA products of 17 and 18 nt in length appeared early during the timecourse and were no longer detectable by ∼ 2 min (Fig. 1b). We also con-ducted similar transcription assays in which the RNA was end-labeled(Fig. 1c). With this labeling protocol, we no longer observed the

smaller (10–17 nt) RNA products that appeared late in the time coursewith the placUV5 template (compare lanes 10–12 in Fig. 1b with lanes9 and 10 in Fig. 1c), suggesting that these RNAs are cleavage productsderived from an internal portion of the RNA.

Next, we tested whether altering the –10-like sequence element inthe initially transcribed region of plac would influence the productionof the 17- and 18-nt RNAs. To do this, we introduced single-base-pairsubstitutions into the placUV5 template that either decreased(A(+2)G) or increased (T(+4)A) the match between the sequencefrom +1 to +6 and a consensus promoter –10 element. When assayswere done using a placUV5 template containing the single-base-pairsubstitution A(+2)G, we no longer detected the 17- and 18-nt RNAspecies (Fig. 2a, lanes 6–10). Similar results were obtained when thebase pair at position +6 was mutated (T(+6)G), also decreasing thematch to a promoter –10 element (data not shown). Conversely, whenthe template bore the T(+4)A substitution, the ratio of the 17- and 18-nt RNA species to the runoff transcript increased about two-fold (Fig. 2a, compare lanes 1–3 with lanes 11–13). These data

Figure 1 In vitro transcription assays with λPR′ and plac templates. (a) DNAsequences in the initially transcribed regions of λPR′ and plac resemble apromoter –10 element. Shown are a consensus extended –10 promoterelement and sequence extending from –6 to +9 of λPR′ and plac. Basesmatching the –10 element in the pause-inducing sequence at λPR′ and theputative pause-inducing sequence at plac are highlighted. (b) Single-roundin vitro transcription assays using either a λPR′ (lanes 1–6) or a placUV5(lanes 7–12) template. RNA was labeled internally with α-32P UTP.Reactions were stopped at the indicated times after transcription wasinitiated. +16 and +17, 16- and 17-nt RNA species, respectively, producedduring assays using the λPR′ template; cleavage products, products thatarose late in the time course using the placUV5 template; T, 194-ntterminated transcript produced on the λPR′ template; RO, 96-nt runofftranscript produced from the placUV5 template. (c) Assays as described in bexcept RNA was end-labeled using γ-32P ATP.

Figure 2 Sequence element in the initially transcribed region of placmediates a σ70-dependent pause in vitro. (a) Single-round in vitrotranscription assays using placUV5-derived templates. RNA was labeledinternally with α-32P UTP. Reactions were stopped at the indicated timesafter transcription was initiated. +17 and +18, 17- and 18-nt RNA species,respectively, produced early during the time course; RO, 96-nt runofftranscript. Reactions were done using either an unmodified placUV5template (lanes 1–5), a template containing the single-base-pairsubstitution A(+2)G (lanes 6–10) or a template containing the single-base-pair substitution T(+4)A (lanes 11–15). (b) Single-round in vitrotranscription assays using a placUV5 template with wild-type or mutant σ70.Assays were done using holoenzyme reconstituted with wild-type σ70

(lanes 1–6) or σ70 L402F (lanes 7–12). RNA was labeled internally with α-32P UTP. Reactions were stopped at the indicated times after transcriptionwas initiated.

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demonstrate that the production of the 17- and 18-nt RNA speciesdepends on the identity of the DNA base pairs in the initially tran-scribed region of the lac promoter (from +1 to +6). Specifically, weobserved a correlation between the degree of resemblance of thissequence to a promoter –10 element and the production of the 17- and18-nt RNA species. A previous analysis of RNAs produced duringtranscription from the lacUV5 promoter revealed RNA products of 17and 18 nt in length that were attributed to pausing24 and probablyarose from the sequence element identified here.

A �70 mutant abolishes the plac pauseA previously isolated amino acid substitution in σ70, L402F, impairsthe ability of RNAP to pause at λPR′ (ref. 23). To assess whether or notthe L402F substitution would also affect formation of the 17- and 18-nt RNA species at placUV5, we conducted transcription assaysusing RNAP holoenzyme reconstituted with either wild-type σ70 orσ70 L402F. When the transcription assays were done using holoenzymereconstituted with σ70 L402F, we no longer observed the 17- and 18-ntRNA species (Fig. 2b). We thus observed a correlation between theeffect of the L402F substitution on the λPR′ pause23 and its effect on theproduction of the 17- and 18-nt RNAs at placUV5. Taken together, theresults of our transcription assays suggest that the 17- and 18-nt RNAspecies produced from the placUV5 template are the result of a σ70-dependent pause that is mediated by a –10-like DNA sequence element in the initially transcribed region of the lac promoter. Underthe conditions of our in vitro analysis (see Fig. 1c), pausing at plac isless efficient than at λPR′ with regard to both the fraction of transcrip-tion complexes that pause and the half-life of the paused complex.

The plac pause is sensitive to Gre factorsThe paused elongation complex at λPR′ exists primarily in a back-tracked state (the result of reverse translocation), with the active centerof RNAP positioned ∼ 4 nt upstream of the RNA 3′ end20. The elonga-tion factors GreA and GreB can facilitate the rescue of backtrackedtranscription complexes in vitro by stimulating internal hydrolysis ofthe RNA to generate a new 3′ end properly positioned at the RNAPactive center25,26. Accordingly, the paused elongation complex at λPR′is sensitive to GreA- and GreB-dependent cleavage in vitro20. To fur-ther compare the properties of the paused elongation complexes atλPR′ and plac, we wished to determine whether the σ70-dependentpause at plac was also sensitive to GreA and GreB in vitro. To do this,we carried out transcription assays with the placUV5 template ineither the presence or absence of GreA or GreB (Fig. 3). We found thatthe 17- and 18-nt RNA species disappeared much more rapidly in thepresence of either GreA or GreB, suggesting that the paused elongationcomplexes at plac, like those at λPR′, are predominantly backtracked.

The plac pause sequence element functions in vivoTo test whether the pausing we observed in vitro might have a role inthe in vivo regulation of the lac promoter, we sought to determinewhether the sequence element in the initially transcribed region of thelac promoter could mediate a σ70-dependent pause in vivo. To do this,

we tested whether this sequence element could substitute functionallyfor the pause-inducing sequence element at λPR′ to allow the λQantiterminator to gain access to the transcription complex (see above).To engage the paused elongation complex at λPR′, λQ must bind to aspecific DNA sequence (the QBE) that is positioned between the pro-moter –10 and –35 elements22 (see Fig. 4a). We therefore constructed aλPR′-plac hybrid template in which we replaced the sequence from –6to +23 of λPR′ with the corresponding sequence from plac (Fig. 4a). Asa control, we confirmed that the pausing observed on this λPR′-plachybrid template in vitro resembles the pausing observed on theplacUV5 template (compare Figs. 4b and 1b). Next we wished to testwhether or not λQ could function as an antiterminator for transcrip-tion initiating at this hybrid promoter in vivo. To do this, we fused theλPR′-plac hybrid promoter along with a downstream terminator to alacZ gene and introduced this construct in single copy on an F′ episomeinto a suitable ∆lacZ reporter strain. The level of lacZ gene expressionin this strain is expected to reflect the ability of λQ to function as anantiterminator for transcription initiating at the hybrid promoter.

We found that λQ (provided by a plasmid) stimulated transcriptionof the lacZ reporter gene downstream of the hybrid promoter by a fac-tor of 3 (as compared with a factor of 22 for an otherwise identicalfusion bearing the wild type λPR′ promoter) (Fig. 4c and data notshown). The ability of λQ to stimulate transcription of downstreamgenes at λPR′ depends on the functional integrity of the pause-inducing sequence22. To test whether or not the stimulatory effect ofλQ at the hybrid promoter was dependent on the functional integrityof the plac pause-inducing sequence element, we mutated the con-served base pairs at positions +2 and +6. These substitutions (A(+2)Gand T(+6)A) reduced λQ-stimulated transcription of the lacZreporter gene when tested singly or in combination (Fig. 4c).

Figure 3 The placUV5 paused elongation complex is sensitive to GreB and GreA. Single-round in vitro transcription assays using a placUV5template. Assays were done in the absence (lanes 1–5) or presence of GreB (lanes 6–10) or GreA (lanes 11–15). RNA was end-labeled using γ-32P ATP.Reactions were stopped at the indicated times after transcription wasinitiated. +17 and +18, 17- and 18-nt RNA species, respectively, producedearly during the time course; RO, 96-nt runoff transcript.

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We showed that the paused elongation complex at plac is sensitiveto Gre factor-mediated cleavage in vitro (Fig. 3). Previous analysisindicated that the removal of GreA in vivo increased the half-life ofthe λPR′ pause markedly, whereas removal of GreB alone had no sub-stantial effect on the pause half-life20. We wondered, therefore,whether the half-life of the plac pause would be similarly increasedby the removal of GreA in vivo. An increase in the plac pause half-lifewould be predicted to increase the substrate available for interaction

with DNA-bound λQ at the λPR′-plac hybridpromoter. We therefore introduced a greAnull allele into the reporter stains carryingthe λPR′-plac-lacZ fusions and assayed theeffect of λQ on lacZ transcription. In thecase of the fusion containing the wild-typepause-inducing sequence element, we foundthat removal of GreA protein increased themagnitude of λQ’s stimulatory effect on lacZtranscription (from three-fold to five-fold;Fig. 4c). In contrast, removal of GreA didnot increase the residual effect of λQ ontranscription from the λPR′-plac-lacZfusions with mutations in the pause-inducing sequence element.

The results obtained with the λPR′-plachybrid promoter demonstrate that the placpause-inducing sequence element can substi-tute functionally for the λPR′ pause-inducingsequence element, albeit less efficiently. Inparticular, this sequence element renders thetranscription complex susceptible to modifi-cation by DNA-bound λQ in vivo. In addi-tion, λQ function at the λPR′-plac hybridpromoter is enhanced by the removal of theGreA protein. On the basis of these findings,we conclude that the DNA sequence elementin the initially transcribed region of plac canmediate a Gre-sensitive σ70-dependent pausein vivo.

Effect of plac pause sequence element on�70 retentionPrevious work using fluorescence resonanceenergy transfer (FRET) has shown that atplacUV5 the majority of early transcriptionelongation complexes retain σ70 (70–100%for transcription elongation complexes con-taining 11–15 nt of RNA16,27). Here, usingthe same FRET assay (Fig. 5a), we haveassessed the effect of the pause-inducingsequence element on σ70 retention, analyz-ing, in parallel, placUV5 (Fig. 5b) and aplacUV5 derivative having a substitutionthat disrupts the pause-inducing sequence(A(+2)G; Fig. 5c). Our results indicate that,in a substantial fraction of transcriptionelongation complexes halted at positions11–15, σ70 is retained in the absence of thesequence element, but the stability of inter-actions between σ70 and the remainder ofthe transcription elongation complex is sub-stantially lower in the absence of the

sequence element (t1/2 ≈ 7–8 min versus t1/2 > 30 min; Fig. 5d).Furthermore, for complexes halted after the synthesis of a 13-, 14- or15-nt RNA transcript (RDe,13, RDe,14 and RDe,15), the initial extentof retention of σ70 is substantially lower in the absence of thesequence element (20–50% at 3 min versus 70–80% at 3 min;Fig. 5d). We conclude that the sequence element considerably stabi-lizes the interaction between σ70 and the remainder of the transcrip-tion elongation complex.

Figure 4 Sequence element in the initially transcribedregion of plac mediates a GreA-sensitive σ-dependentpause in vivo. (a) Top schematic: the presence of λQallows RNAP that has initiated transcription at λPR′ toread through a transcription terminator. Close-up view:functionally important DNA elements at λPR′ includingthe promoter –10 and –35 elements, the Q-bindingelement (QBE) and the pause-inducing sequence. Alsoshown is the sequence of the λPR′-plac hybrid promoter.In this artificial promoter the sequence extending from –6 to +23 of λPR′ has been replaced with thecorresponding sequence from plac (open box).(b) Single-round in vitro transcription assays using eithera wild-type λPR′ template (lanes 1–5) or the λPR′-plachybrid template (lanes 6–10). RNA was labeledinternally with α-32P UTP. Reactions were stopped at theindicated times after transcription was initiated. +16and +17, 16- and 17-nt RNA species, respectively,produced during assays using the wild-type λPR′template; T, 194-nt terminated transcript. (c) Effect of λQ on transcription in vivo from λPR′-plac hybridpromoters. Cells containing the indicated reporter (lacZ fused to a λPR′-plac derivative) on an F′ episomewere transformed with a plasmid that did or did notencode λQ and assayed for β-galactosidase. Shown is the fold stimulation of lacZ expression mediated by

λQ. Indicated above each bar (x/y) are the absolute units of β-galactosidase activity obtained in the presence (x) or absence (y) of λQ. The cells contained a functional greA gene (+) or not (–), asindicated.

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DISCUSSIONWe have shown that a sequence element in the initially transcribedregion of the lac promoter can induce a pause in early elongation. Likea similar sequence element present in the initially transcribed regionof λPR′, the plac pause-inducing sequence element resembles a pro-moter –10 element and mediates formation of a paused elongationcomplex by binding σ70. These findings are consistent with the resultsof an independent study in which the σ70 subunit was found to be pre-sent in a paused elongation complex induced by the plac initially tran-scribed sequence in vitro28.

Our findings suggest that the –10-like sequence element in the placinitially transcribed region may contribute to the regulation of lactranscription. In particular, our demonstration that the –10-likesequence element can induce a σ70-dependent pause in vivo supportsthis possibility. Furthermore, a promoter-proximal pause has beendetected at plac in a ∆greA strain by in vivo footprinting (A. Hatoumand J.W. Roberts, personal communication). However, examination ofeffects of the pause-inducing sequence element on lac gene expressionis complicated by the fact that mutations in this region may affect RNAstructure and/or the binding of the Lac repressor to the lac operator,which overlaps the pause-inducing sequence element, or may haveother unintended consequences29.

Pause-inducing sequence element affects � retentionOur FRET experiments established that σ70 was retained in transcrip-tion elongation complexes even when the pause-inducing sequencewas mutated (A(+2)G), but that the half-life of σ70 retention was

reduced (more than four-fold) under these circumstances (Fig. 5d). Incontrast, σ70 was not detected by Brodolin et al.28 in stalled elongationcomplexes prepared at placUV5 when the pause-inducing sequenceelement was mutated (T(+6)A). We emphasize that our FRET experi-ments did not involve a separation step (that is, no chromatography,electrophoresis, sedimentation or washing) or long incubations (noincubations >10 min). The inclusion of a separation step and/or a longincubation disrupts interactions between σ70 and the remainder of thetranscription elongation complex—especially the weak interactions inthe absence of the pause-inducing sequence element—and results,artifactually, in loss of σ70 (ref. 16; J.M. and R.H.E., unpublished data).We suggest that the inclusion of a separation step by Brodolin et al.28

accounts for the fact that these investigators did not detect σ70 installed elongation complexes when the pause-inducing sequence ele-ment was mutated.

Our observation that the initial extent of σ70 retention was reducedwhen the pause-inducing sequence element was mutated (from ∼ 70%to ∼ 20% for RDe,15, for example) raises the possibility that sequenceelements that promote σ70-dependent pauses may influence the σ70

content of elongation complexes downstream of the pause site. In par-ticular, we suggest that the ability of a σ70-dependent pause-inducingsequence element to increase the initial extent of σ70 retention couldincrease the proportion of elongation complexes containing σ70

downstream of the pause. In support of this proposal, it has beenshown that, at placUV5, ∼ 60% to 70% of transcription elongationcomplexes containing a 50-nt transcript retain σ70 when the pause-inducing sequence element is intact16.

Figure 5 Sequence element in the initiallytranscribed region of plac stabilizes interactionsbetween σ70 and the remainder of thetranscription elongation complex. (a) Use of σ70-DNA FRET to assess retention of σ70 intranscription elongation complexes16,27. D, donorfluorochrome incorporated in σ70; A, acceptorfluorochrome incorporated in downstream DNA.(b) DNA fragments used in analysis oftranscription elongation complexes initiated atplacUV5 and halted after synthesis of 11, 12,13, 14 and 15 nt of RNA (that is, lacUV5derivatives having no guanine residues on thetemplate strand from position +1 to +11, +1 to+12, +1 to +13, +1 to +14, or +1 to +15, andhaving Cy5 at position +25, +26, +27, +28 or+29. Black boxes, transcription start site (witharrow), promoter –10 element and promoter –35element; red boxes, halt site. (c) DNA fragmentsused in analysis of transcription elongationcomplexes initiated at placUV5 derivative havinga single-base-pair substitution that disrupts thesequence element (placUV5(A(+2)G)) and haltedafter synthesis of 11, 12, 13, 14 and 15 nt ofRNA. Black boxes, transcription start site (witharrow), promoter –10 element and promoter –35 element; blue boxes, A(+2)G substitution;red boxes, halt site. (d) Retention of σ70 intranscription elongation complexes containing11, 12, 13, 14 or 15 nt of RNA (RDe,11, RDe,12,RDe,13, RDe,14 and RDe,15) determined by σ70-DNA FRET. Circles, transcription elongationcomplexes initiated at placUV5; triangles,transcription elongation complexes initiated atplacUV5(A(+2)G). Data are means ± s.d. of twoor three independent determinations.

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Pause-inducing sequences at other promotersThe demonstration that a –10-like sequence element in the initiallytranscribed region of plac, like the sequence element in the initiallytranscribed region of λPR′, can induce σ70-dependent promoter-proximal pausing indicates that this phenomenon does not depend onunique features of the late promoters of the lambdoid phages. Thepause-inducing sequence elements at plac and λPR′ consist of: (i) a 3/6match to the –10-element consensus sequence, TATAAT, in register +1(where the register is defined by the first base of the –10-like hexamer)(ii) an A at position +2 and (iii) a T at position +6 (though sequencedeterminants flanking the element can affect pausing30). To determinethe frequency of occurrence of these three features in σ70-dependentpromoters, we analyzed a database of sequences of known E. coli σ70-dependent promoters (652 promoters31). Of the 652 σ70-dependent promoters analyzed, 5.1% (33 promoters) containan equal or better (≥3/6) match to TATAAT in register +1, an A at position +2 and a T at position +6 (Table 1). Of these 33 promoters, 12contain a better (≥4/6) match to TATAAT, and 5 contain a substantiallybetter (≥5/6) match to TATAAT (Table 1).

In the late promoters of lambdoid phages, the pause-inducingsequence element can be located in register +1 through at least +9 (+1in λPR′; +2 in phage 21 PR′ and phage 80 PR′; +9 in phage 82 PR′;ref. 14). Inspection of the database of sequences of known E. coli σ70-dependent promoters indicates fully 17% (112 promoters) con-tain a ≥4/6 match to TATAAT in registers +1 through +9, with an A atthe second position and a T at the sixth position (Table 1). Of these112 promoters, 25 contain a ≥5/6 match to TATAAT and 1 promotercontains a perfect 6/6 match to TATAAT (Table 1). The analysis indi-cates that a substantial fraction of promoters contain initially tran-scribed region –10-like elements that are equivalent, or in fact bettermatches to the consensus that those at λPR′ and plac. We suggest thatσ70-dependent promoter proximal pausing is likely to occur at many,if not most, of these promoters.

We propose that σ70-dependent pause-inducing sequence elementsmight function to limit downstream gene expression, an effect thatcould be modulated by cellular factors such as the Gre proteins. In par-ticular, the presence of a promoter-proximal pause-inducing sequenceelement would be predicted to increase the spacing between RNAPmolecules on the transcription unit downstream of the pause. An increase in the spacing between RNAP molecules within a tran-scription unit has been shown to affect the ability of RNAP to pause orterminate at promoter-distal locations32,33. Therefore, promoter-proximal σ70-dependent pauses could, in principle, affect events

occurring further downstream by influencing both σ retention inindividual transcription complexes and RNAP density within a tran-scription unit.

METHODSExpression vectors. Plasmid pBRλQ (used in Fig. 4c) harbors the wild-type λQgene on an NdeI-BamHI fragment under the control of the lacUV5 promoter34.Plasmid pBR∆Q (used in Fig. 4c) encodes no functional λQ (ref. 34).

Test promoters and reporter strains. Reporter strain BN147 contains sequenceextending from –109 to +232 of λPR′ fused to a lacZ reporter gene. Thisreporter fusion is present on an F′ episome and has been described34. Thesequence between –6 and +23 of λPR′ was replaced with the correspondingsequence from plac to create the artificial promoter λPR′-plac. Derivatives ofλPR′-plac that carry base pair substitutions A(+2)G, T(+6)A, or A(+2)GT(+6)A were constructed as well. Each of these promoters was cloned on anEcoRI-HindIII-cut PCR product into pFW11 Tet34. These pFW11 plasmidswere used to create the following reporter strains that carry the λPR′-plac deriv-atives fused to lacZ, as described35: BN343 containing λPR′-plac, BN401 con-taining λPR′-plac T(+6)A, BN412 containing λPR′-plac A(+2)G, and BN416containing λPR′-plac A(+2)G T(+6)A.

We constructed a derivative of strain FW102 (ref. 35) that does not produceany functional GreA. This strain, BN105, is essentially FW102 greA::CM. P1transduction was used to transfer a greA::CM allele from strain JWR1018(ref. 20) to strain FW102. Strains BN343, BN401, BN412 or BN416 (see above)were mated with strain BN105 to create the following reporter strains that didnot produce any functional GreA: BN426 (λPR′-plac), BN427 (λPR′-placT(+6)A), BN428 (λPR′-plac A(+2)G) and BN429 (λPR′-plac A(+2)G, T(+6)A).

Assay of �-galactosidase activity. Cells were grown in LB supplemented withcarbenicillin (100 µg ml–1) and tetracycline (10 µg ml–1). IPTG was provided at200 µM, a concentration sufficient to induce maximal expression of λQ, and toprevent the binding of Lac repressor (encoded by the F′ episome) to the lacoperator present in the initially transcribed region of the λPR′-plac hybrid pro-moter. SDS-CHCl3 permeabilized cells were assayed for β-galactosidase activityessentially as described36. Assays were done at least three times in duplicate onseparate occasions, with similar results. Values are the averages from one exper-iment; duplicate measurements differed by <5%.

In vitro transcription. Open complexes were formed at λPR′ or λPR′-plac byincubating 20 nM RNAP with 5 nM template for 5 min at 37 °C in transcriptionbuffer (20 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 10 mM DTT, 50 mM KCl and100 µg ml–1 BSA) plus 200 µM ATP, GTP and CTP and 50 µM UTP. Open com-plexes were formed at placUV5 (or placUV5 derivatives) by incubating 50 nMRNAP with 20 nM template for 10 min at 37 °C in transcription buffer plus200 µM ATP, GTP and CTP and 50 µM UTP. Assays were done in the presenceof 0.5 µC µl–1 α-32P UTP (results in Figs. 1b, 2a,b and 4b) or 1 µC µl–1 γ-32PATP (results in Figs. 1c and 3). A single round of transcription was initiated byadding 4 mM MgCl2 and 10 µg ml–1 rifampicin. Where present, GreA or GreBwas added to 250 nM at 90 s before the addition of the MgCl2-rifampicin mix-ture. The final volume of each reaction was 150 µl. At the indicated times afterthe addition of the MgCl2-rifampicin mixture, 20 µl of each reaction wasremoved and quenched as described23. Samples were then extracted with phe-nol-chloroform (1:1), precipitated with ethanol, resuspended in 4 µl of loadingbuffer (95% (v/v) formamide, 20 mM EDTA, 0.05% (w/v) bromophenol blue,0.05% (w/v) xylene cyanol), and electrophoresed on 15% (w/v) polyacrylamidesequencing gels. Gels were visualized by phosphorimager.

Linear templates for the in vitro transcription assays done with the λPR′-based templates were made by the PCR from pFW11-derived plasmidscarrying sequence extending from –109 to +232 of either λPR′ or λPR′-plac.Linear templates for assays done with the placUV5-based templates were madeby the PCR from pFW11 Tet-derived plasmids carrying sequence extendingfrom –60 to +38 of placUV5. Single-base-pair substitutions A(+2)G andT(+4)A were introduced into placUV5 by the PCR.

FRET experiments. Preparation of RNAP derivatives (RNAP holoenzyme and RNAP holoenzyme labeled at position 366 of σ70), preparation of DNA

Table 1 Analysis of known �70-dependent promoters for potentialpause-inducing sequence elements

NANNNT and ≥3/6 match to TATAAT in register 1: 33a (5.1%)

NANNNT and ≥4/6 match to TATAAT in register 1: 12b (1.8%)

NANNNT and ≥5/6 match to TATAAT in register 1: 5c (0.77%)

NANNNT and 6/6 match to TATAAT in register 1: 0 (0%)

NANNNT and ≥4/6 match to TATAAT in registers 1–9: 112 (17%)

NANNNT and ≥5/6 match to TATAAT in registers 1–9: 25 (3.8%)

NANNNT and 6/6 match to TATAAT in registers 1–9: 1d (0.15%)

Sequences of promoters were provided by A. Huerta and J. Collado-Vides, UniversidadNacional Autónoma de México (for sequences seehttp://www.cifn.unam.mx/Computational_Genomics/regulondb/).afolA, ilvIp3, codB, lac, mhpA, fepA, fes, entC, sdhp2, fiup, ycbP, topAp3, nohAp, malX, purR,infCp2, asnU, gnd, micFp1, fabB, ptsHp0, cysP, upp, emrR, speB, speCp1, rpoDp2, crp, slp,cspAp2, ivbL, metB, metH. bilvIp3, mhpA, fes, entC, fiup, nohAp, purR, micFp1, ptsHp0,speCp1, rpoDp2, slp. cfes, nohAp, micFp1, speCp1, rpoDp2. drplJ.

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A R T I C L E S

550 VOLUME 11 NUMBER 6 JUNE 2004 NATURE STRUCTURAL & MOLECULAR BIOLOGY

fragments, measurement of leading-edge FRET and trailing-edge FRET (in-gel), and data reduction were done as described16,27, except that both lacUV5and lacUV5(A(+2)G) derivatives were analyzed, and that ApA was replaced byApG in analysis of lacUV5(A(+2)G) derivatives.

Proteins. His-tagged versions of wild-type σ70 and σ70 L402F were purified asdescribed after overproduction from plasmid pLHN12-His37. E. coli RNAPcore was obtained from Epicentre, and holoenzyme was made by incubationwith a five-fold excess of the appropriate σ70. Purified GreA and GreB were pro-vided by S. Borukhov.

ACKNOWLEDGMENTSWe thank J. Roberts, K. Brodolin, A. Kapanidis and S. Weiss for discussion, S. Borukhov for purified GreA and GreB, A. Huerta and J. Collado-Vides forproviding promoter sequences, S. Dove for comments on the manuscript, and R. Hellmiss for assistance with figure preparation. We also thank A. Hatoum and J. Roberts for sharing unpublished results. This work was supported by USNational Institutes of Health grant GM44025 to A.H. and GM41376 to R.H.E. and by a Howard Hughes Medical Institute investigatorship to R.H.E.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 8 December 2003; accepted 15 March 2004Published online at http://www.nature.com/nsmb/

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