Interaction of Escherichia coli RNA Polymerase σ70 Subunit with Promoter Elements in the Context of Free σ70, RNA Polymerase Holoenzyme, and the β′-σ70 Complex*

Promoter recognition by RNA polymerase is a key point in gene expression and a target of regulation. Bacterial RNA polymerase binds promoters in the form of the holoenzyme, with the σ specificity subunit being primarily responsible for promoter recognition. Free σ, however, does not recognize promoter DNA, and it has been proposed that the intrinsic DNA binding ability is masked in free σ but becomes unmasked in the holoenzyme. Here, we use a newly developed fluorescent assay to quantitatively study the interactions of free σ70 from Escherichia coli, the β′-σ complex, and the σ70 RNA polymerase (RNAP) holoenzyme with non-template strand of the open promoter complex transcription bubble in the context of model non-template oligonucleotides and fork junction templates. We show that σ70, free or in the context of the holoenzyme, recognizes the −10 promoter element with the same efficiency and specificity. The result implies that there is no need to invoke a conformational change in σ for recognition of the −10 element in the single-stranded form. In the holoenzyme, weak but specific interactions of σ are increased by contacts with DNA downstream of the −10 element. We further show that region 1 of σ70 is required for stronger interaction with non-template oligonucleotides in the holoenzyme but not in free σ. Finally, we show that binding of the β′ RNAP subunit is sufficient to allow specific recognition of the TG motif of the extended −10 promoter element by σ70. The new fluorescent assay, which we call a protein beacon assay, will be instrumental in quantitative dissection of fine details of RNAP interactions with promoters.

Bacterial RNA polymerase (RNAP) 3 initiates transcription in the form of the holoenzyme (subunit composition ␣ 2 ␤␤Ј). The RNAP core enzyme (␣ 2 ␤␤Ј) is catalytically competent but does not recognize promoters. Available biochemical, structural, and genetic data clearly show that, within the holoenzyme, is responsible for the recognition of promoter consensus elements located, at most promoters, ϳ10 and ϳ35 bp upstream of the transcription start site (located at ϩ1) (1)(2)(3). Although some promoters lack the Ϫ35 element (and hence do not require region 4 for promoter complex formation), the Ϫ10 promoter element is strictly required. However, the Ϫ10 promoter element on its own is not sufficient for promoter complex formation. In the absence of the Ϫ35 element, additional elements such as a TGX motif upstream of the Ϫ10 element or downstream GGGA motif are required for efficient promoter recognition (4,5).
The Ϫ10 promoter element is recognized by region 2. Although original recognition must proceed in the form of double-stranded DNA, in transcription-competent open complex, the Ϫ10 element is present in single-stranded form as part of the transcription bubble, which on most promoters extends from Ϫ12 to approximately ϩ3. Although open complex formation by RNAPs from mesophilic organisms such as Escherichia coli takes place under conditions at which the double-stranded form of DNA is resistant to denaturation by about 1 kcal/mol bp, many open complexes are very stable (6). Because of high energetic cost of transcription bubble formation, the RNAP-promoter interactions responsible for this process must be very strong. An important source of energy driving the strand separation process and controlling the open complex stability is the interaction of RNAP with the non-template strand of the transcription bubble (6 -8).
In the open complex, region 2 makes specific contacts with the non-template strand of the Ϫ10 promoter element (consensus sequence TATAAT for E. coli 70 as well as for housekeeping factors of many other bacteria). Adenine at position Ϫ11 of the non-template strand is of special importance for nucleation of promoter melting (9). Outside of the Ϫ10 element, a non-template segment of the transcription bubble corresponding to positions Ϫ6 to Ϫ3 interacts with region 1.2 (10) and cross-links to the RNAP ␤ subunit (11). Further downstream, a non-template segment of the transcription bubble surrounding the transcription start point (positions Ϫ2 to ϩ2) interacts with ␤ (11). Gralla and coworkers (12) showed that inclusion of the non-template segment corresponding to positions Ϫ6 to ϩ1 stimulates the binding of fork junction probes, which consist of doublestranded upstream promoter DNA with single-stranded se-quences downstream of positions Ϫ11 or Ϫ12. It is unknown, however, whether such increased binding is sufficient to mediate formation of the transcription bubble.
Specific interaction of oligonucleotides mimicking the nontemplate strand of the transcription bubble in the open promoter complex (i.e. containing the Ϫ10 promoter element and downstream DNA up to position ϩ1) with is readily detected using biochemical methods such as protein-DNA cross-linking or native gel electrophoresis in the context of the holoenzyme (8). In fact, isolated ␤Ј or a ␤Ј fragment containing the primary -binding site is sufficient to allow relatively strong interaction between (or fragment containing region 2) with such oligonucleotides (13,14). These results were interpreted as suggesting that, upon formation of the holoenzyme, the intrinsic Ϫ10 element binding capacity of is either unmasked (by removing an inhibitory interaction that is present in free and that prevents promoter recognition) or increased (by introducing a conformational change and/or readjusting the DNA binding domain of ). Indeed, it is now well established that does undergo large scale conformational changes upon holoenzyme formation that affect the relative positions of its DNA binding domains (15,16).
In this study, we use a new fluorometric assay to monitor site-specific interactions of promoter DNA and its fragments with holo-RNAP, free 70 , and with the ␤Ј-70 complex. Using this new method, we dissect the energetics of RNAP interaction with the non-template segment of the transcription bubble in the context of oligonucleotide and fork junction model promoter fragments. The results indicate that RNAP binding to the Ϫ10 element and to the downstream Ϫ6/Ϫ3 fragment is highly energetically favorable and can provide a major part of energy required for local promoter melting, whereas interaction with the Ϫ2/ϩ2 segment is relatively weak. We show further that formation of the holoenzyme increases the apparent affinity of oligonucleotides mimicking the non-template strand of the transcription bubble by ϳ300fold. In contrast, formation of the holoenzyme has only a modest effect on the interaction of with shorter non-template oligonucleotides corresponding to the Ϫ10 promoter element alone.
Plasmids-Plasmid pGEMD(ϪCys) encoding a 70 derivative with no Cys residues and plasmid encoding 70 derivative with single Cys residue at position 211 were described previously (17,18). Plasmid encoding a 70 derivative with a single Cys residue at position 192 and a plasmid encoding a 70 derivative with a single Cys residue at position 211 combined with W434A,W433A substitutions was constructed from plasmid pGEMD(ϪCys) by site-directed mutagenesis. Plasmid for overproduction of 70 (104 -613) derivative with a single Cys residue at position 211 was constructed by amplify-ing a DNA fragment encoding amino acids 104 -613 of the 70 derivative with appropriate primers introducing NdeI and EcoRI restriction sites at the 5Ј and 3Ј ends of the fragment, respectively, and cloning the amplified fragment between the NdeI and EcoRI sites of the pET28a expression vector.
Protein Purification and Labeling-Single Cys 70 derivatives were prepared as in Ref. 17. Fluorescent labels were incorporated into single Cys 70 derivatives using Cys-specific chemical modification (procedures as described in Ref. 19), and efficiencies of labeling were Ͼ70%. Labeled RNAP holoenzyme derivatives were reconstituted by mixing the RNAP core and labeled 70 at a ratio of 1 DNA Probes-Promoter DNA fragments were formed by mixing equimolar amounts of synthetic complementary strands in a buffer containing 40 mM Tris, pH 7.9, 100 mM NaCl, heating for 2 min at 95°C, and slowly cooling down to 25°C.
Fluorometric Assays-Fluorescence measurements were performed using a Quanta-Master QM4 spectrofluorometer (PTI) in transcription buffer (TB: 40 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5% glycerol, 1 mM DTT, and 10 mM MgCl 2 ) containing 0.02% Tween 20 at 25°C. Final assay mixtures (800 l) contained 1 nM labeled 70 or RNAP holoenzyme and DNA probes at various concentrations. The (␤Ј-70 ) complex assay mixtures contained 1 nM (211Cys-TMR) 70 and 30 nM ␤Ј. The 30 nM ␤Ј concentration saturated the fluorescence signal increase in the experiments. Some control experiments were performed using 3 nM labeled free 70 or RNAP holoenzyme. We did not observe a difference between the K d values measured at either 1 or 3 nM of a beacon concentration. The TMR fluorescence intensities were recorded with an excitation wavelength of 550 nm and an emission wavelength of 578 nm. Kinetic measurements were performed as in Ref. 23.
Data Analysis-To obtain equilibrium dissociation constants (K d ), the experimental dependence of the fluorescent signal amplitude (F) on DNA probe concentration (C) was fit to Equation 1, ; F o is the initial value of the amplitude, and F max is the limiting value of the amplitude at C ϭ ∞.
Oligo probes bearing mutations at Ϫ7 and Ϫ11 positions produced very low fluorescent signal. Dissociation constants of these probes were obtained from competition binding experiments using the consensus single-stranded probe as a ref-erence. The experiments were carried out at a fixed concentration of a substituted probe and various concentrations of consensus probe. The data were fit to Equation 2, where K d,0 and C 0 are dissociation constant and concentration of the substituted probe, respectively. Equation 2 is valid at C 0 Ͼ Ͼ [RNAP], a condition that was fulfilled in our experiments.

RESULTS
Design of a Protein Beacon Assay-Tryptophan and tyrosine side chains quench fluorescence of adjacent organic fluorophores (dyes) due to photoinduced electron transfer (PET) between the first excited singlet state of the dye and the ground state of Trp or Tyr (26). Efficient PET usually occurs at length scales below 1 nm. We sought to develop a simple PET-based, one-label quantitative assay to monitor the interaction of bacterial RNAP with promoter DNA. We reasoned that the interaction of the E. coli RNAP 70 subunit region 2 with the Ϫ10 promoter element might lend itself to such an assay because it is known that this interaction involves multiple aromatic amino acids of 70 region 2.3 that change their environment upon interaction with DNA (27)(28)(29). The exact nature of this change is, however, unknown.
Schematic representation of the assay is shown in Fig. 1A. The assay is based on measuring emission from a fluorescent label attached to E. coli 70 in the vicinity of a cluster of Trp and Tyr residues from conserved region 2.3 (Trp-433, Trp-434, Tyr-425, and Tyr-430). The base-line fluorescence of labeled free 70 or RNAP holoenzyme that contains it is expected to be low due to quenching by the nearby tryptophan and tyrosine residues. Upon DNA binding and establishment of specific interactions with nucleotides from the non-template strand of the Ϫ10 promoter element, the contacts between the fluorescent label and residues from the aromatic cluster of region 2.3 can become disrupted, which should result in decreased quenching and the enhancement of the fluorescent signal. The assay reports only on protein-DNA interactions confined to small parts of RNAP and DNA adja-cent to 70 region 2.3 aromatic residues, i.e. on specific interactions important for promoter complex formation. The assay thus should be "blind" to interactions that occur elsewhere in the RNAP molecule. By analogy with previously described molecular and peptide beacons assays (30,31), we name the new assay a protein (RNAP) beacon assay.
Development and Validation of a Protein Beacon Assay-Several previously described functional single cysteine mutants of E. coli 70 that allow site-specific introduction of fluorescent labels were prepared. The mutants were chosen based on spatial proximity of modified residues to 70 region 2.3 in available structures containing subunits from E. coli and Thermus spp. (3,32). A structural model of (211Cys-TMR) 70 , 70 labeled at position 211 with fluorescent label, 5-TMR, is shown in Fig. 1B as an example.
RNAP holoenzymes reconstituted from mutant labeled with 5-TMR, BODIPY FL, or ATTO-520 (the efficiency of labeling was at least 70%) were functional (at least 80% of the wild-type RNAP activity in abortive initiation assay on the T5N25 promoter). Reconstituted fluorescently labeled RNAP holoenzymes were combined with a double-stranded DNA fragment containing the T5N25 promoter (shown in Fig. 2A), and changes in fluorescence were monitored. Upon addition of promoter DNA to RNAP holoenzymes containing mutant with 5-TMR attached to residue 192 or 5-TMR, BODIPY FL, or ATTO-520 attached to residue 211, the fluorescent signal increased ϳ5-fold and saturated in a few minutes. A typical trace, obtained with RNAP holoenzyme containing (211Cys-TMR) 70 and a DNA fragment containing the T5N25 promoter, is shown in Fig. 2B. Similar increases were observed when DNA fragments containing the T7 A1 or lacUV5 promoters were used. Although the interaction between RNAP holoenzyme beacons and promoter-containing DNA fragments was readily detected at 0.5 nM promoter concentration, no signal increase was observed upon mixing free (211Cys-TMR) 70 with 500 nM promoter fragments, indicating, in agreement with previous data, that free 70 is unable to specifically interact with promoters (1,2).
Several control experiments were performed to prove that the interaction detected in the protein beacon assay is specific. In one experiment, the interaction of a 35-nt-long double-stranded "upstream" promoter fragment containing the UP element and the Ϫ35 element but truncated at position Ϫ23 and therefore lacking the Ϫ10 promoter element ( Fig.  2A) with RNAP was studied. Because RNAP elements that can sequence-specifically interact with this fragment are remote from region 2.3, the expectation was that RNAP binding to this promoter fragment should not lead to an increase in the fluorescence signal intensity. Indeed, we found that the addition of 100 nM of upstream fragment to RNAP-(211Cys-TMR) 70 resulted in a fluorescence intensity increase that was 10 times lower than that generated by 2 nM of T5N25 promoter DNA (data not shown). The residual weak fluorescence intensity increase may be caused by nonspecific binding of upstream probe to 70 region 2.3. The rate of signal increase caused by the addition of T5N25 promoter DNA to RNAP-(211Cys-TMR) 70 was considerably slowed down by the presence of 100 nM of the upstream fragment (Fig. 2B), indicating that this fragment bound RNAP and competed for the binding with T5N25 DNA.
Specific interaction of oligonucleotide whose sequence corresponded to positions Ϫ18 to ϩ1 of the phage pRЈ promoter non-template strand with the 70 holoenzyme was previously reported by Marr and Roberts (8), who used biochemical methods (a gel retardation assay) to show that the interaction is characterized by a K d of 3 nM and that a substitution of a T at position Ϫ12 at the upstream end of the Ϫ10 consensus element by a C decreased the interaction 5-fold. We determined dissociation constants for oligonucleotides identical to those used by Marr and Roberts (8) Table S1). Although dissociation constants for "wild-type" oligo C (K d (C)) and mutant oligo M K d (M) varied within a factor of 5 (from 6.2 to 28.8 nM for oligo C) for different beacons, individual K d (M)/K d (C) ratios were close to 4 for every beacon tested (supplemental Table  S1). The ratios of K d values are in good agreement with the Marr and Roberts data (8). The differences in apparent binding constant values observed with individual beacons and in gel retardation assay could be at least partially due to the fact that some part of free energy of oligonucleotide binding must be used to disrupt the van der Waals interaction of the fluorophore with the quencher. If so, then dissociation constants calculated using the beacon assay are expected to be somewhat higher than the actual values for unmodified RNAP, as is indeed observed. An oligo that contained nucleotides most rarely found at each position of the Ϫ10 hexamer (oligo A of Marr and Roberts (8)) did not generate any sig- nal (supplemental Fig. S1). The K d value for oligo A was determined from a competition binding experiment with oligo C as a reference and found to be 15-fold lower than K d for oligo C.
Precise quantification of partial contributions of region 2.3 aromatic residues to fluorescence quenching is complicated by their involvement in the Ϫ10 element binding (27)(28)(29). We found that (211Cys-TMR) 70 holoenzyme carrying a double substitution of Trp-433 and Trp-444 to Ala exhibited less than 10% increased fluorescence in the presence of 200 nM promoter DNA (data not shown). The absence of increased fluorescence was not caused by the inability of the holoenzyme carrying fluorescently labeled W433A,W434A 70 to bind to promoter DNA, as evidenced by gel retardation analysis (data not shown), and is in agreement with the fact that a 70 RNAP mutant bearing four amino acid substitutions W433A, W434A, F427A, and Y430A forms complexes that resemble those formed by wild-type RNAP (33). Trypsin digestion of (211Cys-TMR) 70 enhanced the TMR fluorescence intensity 3.8-fold (data not shown), which can be explained by breaking of TMR contacts with the aromatic residue quenchers. In contrast, similar digestion of (211Cys-TMR, W433A,W434A) 70 resulted in only 8% signal increase. These results indicate that Trp-433 and Trp-434 indeed play an important role in the 211Cys-TMR fluorescence quenching.
If increased fluorescence observed in the presence of promoter DNA were due to formation of promoter complexes, then a decrease in fluorescence intensity should be observed upon RNAP escape from promoter into elongation. To test this prediction, nucleotide triphosphates and unlabeled 70 (10-fold excess as compared with the labeled 70 protein) were added to preincubated reactions containing fluorescent RNAP holoenzyme and promoter DNA (Fig. 2C). Unlabeled 70 was added to prevent recapturing of dissociated fluorescently labeled by the core, which could have led to reformation of promoter complexes with fluorescently labeled holoenzyme. As can be seen, fluorescence decreased upon the addition of nucleotide triphosphates and unlabeled by 2.5fold. This effect was not observed in the presence of 1 M Rifampicin, a drug that prevents RNAP escape from promoter. We interpret this result as suggesting that a decrease in fluorescence in the absence of rifampicin is due to RNAP leaving the promoter, which leads to disruption of contacts with DNA.
Based on the data presented above, we conclude that our PET-based protein beacon assay is behaving as expected from design and that RNAP holoenzymes containing fluorescently labeled 70 provide a simple, quantitative, and real time assay to measure RNAP interactions with promoter DNA.
In what follows, we use the protein beacon assay to study the interaction of model promoter fragments with RNAP holoenzyme, the ␤Ј-70 complex, and free 70 . Data for beacons based on (211Cys-TMR) 70 are presented because these beacons generated the highest signal intensity upon DNA interaction in the context of both RNAP holoenzyme, the ␤Ј-70 complex, and free 70 .
The efficiency of free 70 and RNAP holoenzyme interactions with even shorter oligos corresponding to T5N25 promoter positions Ϫ12/Ϫ3 and Ϫ12/Ϫ6 (Fig. 3A) was determined next (Fig. 3, B-D and Table 2). Corresponding mutant oligos with substitution of T in position Ϫ7 for C were used as controls. For free , the following results were obtained ( Fig.  3D and Table 2). The Ϫ12/Ϫ3 oligo bound as efficiently as the Ϫ12/ϩ2 oligo (K d Ϸ40 M), indicating that non-template DNA nucleotides downstream of position Ϫ3 do not contribute to binding. The binding avidity of the Ϫ12/Ϫ6 oligo was ϳ5-fold lower (K d ϭ 220 M). In all cases, the binding was specific as it was severely affected by substitutions in the Ϫ11 and Ϫ7 positions. The Ϫ12/Ϫ3 and Ϫ12/Ϫ6 oligos bound the holoenzyme with ϳ10 and ϳ500-fold lower affinity than the Ϫ12/ϩ2 oligo (Fig. 3, B and C, and Table 2). The Ϫ12/Ϫ7 oligo, which corresponded exactly to the Ϫ10 promoter element TATAAT, was also tested and was found to bind both free 70 and holo-RNAP poorly; in fact, we could not achieve saturation (data not shown).
It is instructive to compare ratios of dissociation constants for complexes formed by free 70 and RNAP holoenzyme with promoter oligos of the same length ( Table 2). The shortest oligo, containing the entire Ϫ10 promoter element and one downstream nucleotide, oligo Ϫ12/Ϫ6, binds the holoenzyme only 2.8 times better than free 70 . An oligo of intermediate length (Ϫ12/Ϫ3) binds the holoenzyme 29 times better than free 70 . The longest oligo of the set, the Ϫ12/ϩ2 oligo, binds ϳ300 times more avidly to RNAP holoenzyme than to 70 . In other words, the shorter the oligo containing the Ϫ10 promoter element sequence, the less the difference between the efficiency of its binding to free and the holoenzyme. This result suggests that the much stronger binding of the Ϫ12/ϩ2 oligo to the holoenzyme than to 70 mainly results from interactions outside of the Ϫ10 element and may involve RNAP core subunits or regions of 70 other than region 2.
Interactions of RNAP Holoenzyme and Free 70 with Fork Junction Probes-Holo-RNAP specifically binds fork junction DNAs, model promoter substrates containing doublestranded upstream DNA, and single-stranded extensions corresponding to the non-template strand of the Ϫ10 promoter element (34). We evaluated free energy (⌬G) of RNAP inter-action with the Ϫ12/Ϫ7, Ϫ6/Ϫ3, and Ϫ2/ϩ2 segments of the non-template strand of the transcription bubble in the context of fork junction DNA probes based on the T5N25 promoter sequence (shown in Fig. 4). The RNAP interactions with the Ϫ2/ϩ2 segment were also studied using fork junction probes based on the lacUV5 promoter sequence. The ⌬G Ϫ12/Ϫ7 ,⌬G Ϫ6/Ϫ3 , and ⌬G Ϫ2/ϩ2 values were quantified by comparing K d values of a fork junction probe bearing a singlestranded segment of interest with a K d values of a similar probe that lacked such a segment. Such measurements could not be carried out using probes with identical upstream double-stranded fragments because K d values for probes with the shortest and longest single-stranded segments differ more than 10 7 -fold. Therefore, probe affinities were adjusted by making changes in the sequence and/or shortening the double-stranded segment.
RNAP binding to several fork junctions was found to be very strong (K d Ͻ0.1 nM). We measured the K d value of such tight complexes using an equilibrium competition binding assay. A double-stranded model promoter fragment containing the consensus UP element, the Ϫ35 element, and the TG motif of the extended Ϫ10 element was used as a competitor ((Ϫ58/Ϫ14) probe, shown in supplemental Fig. S3A). Upon binding to the holo-RNAP beacon, the (Ϫ58/Ϫ14) probe generates a negligible signal. Yet the binding of this probe is very tight (K d ϭ 0.015 nM) as revealed by a competition binding experiments using the (Ϫ58/Ϫ14) probe and other probes, for which K d values were determined by titration.
Equilibrium dissociation constants of RNAP holoenzyme complexes with the set of probes described above are shown in Table 3. As can be seen, the Ϫ7/Ϫ12 single-stranded segment increased the binding to RNAP by ϳ30,000-fold. A fork junction containing a single-stranded extension up to position Ϫ3 bound ϳ200-fold better than the corresponding probe extending to position Ϫ7. Finally, extension of the singlestranded segment to position ϩ2 increased the binding efficiency ϳ10-fold, compared with the template extending to position Ϫ3 (the latter result was obtained on both T5N25 and lacUV5-based probes). We conclude that the data on dissection of the energetics of RNAP interaction with the transcription bubble non-template stand obtained with the oligonucleotide and fork junction model promoter fragments indicate that RNAP binding to the to the Ϫ10 element and to a segment immediately adjacent to the Ϫ10 element is quite strong, whereas the downstream segment interacts comparatively weakly.  The binding of free (211Cys-TMR) 70 to fork junction probes was also measured using the protein beacon assay. The fork junctions DNA probes used in these experiments are shown in supplemental Fig. S2A. These probes are identical, but one of them contains a TG motif of the extended Ϫ10 element. The affinities of free (211Cys-TMR) 70 to the fork junction probes were somewhat lower than those found for the Ϫ12/Ϫ3 oligo (K d ϳ100 M; supplemental Fig. S2B). The interaction, however, was specific, because substitutions in the single-stranded part of the fork junction template that decreased the similarity to the Ϫ10 element consensus sequence decreased the binding. The presence of the TG motif had no effect on the binding affinity of (211Cys-TMR) 70 , indicating that free is unable to recognize this element, at least in the context of fork junction probes.
Interactions of the ␤Ј-70 Complex with Model Promoter Fragments-The main site of 70 interaction with the RNAP core enzyme is located in the ␤Ј subunit (35). The interaction is thought to cause conformational changes in 70 that affect its ability to interact with DNA. Thus, Young et al. (14) demonstrated using photocross-linking and LRET techniques that ␤Ј or a short ␤Ј fragment that binds is sufficient to induce detectable binding of to Ϫ18/ϩ1 and Ϫ13/ϩ1 lacUV5 nontemplate strand oligos. The binding of isolated 70 to oligos was not detectable by the method used. We measured K d val-ues for the binding of our set of Ϫ10 oligos as well as the lacUV5 Ϫ18/ϩ1 non-template strand oligo used by Young et al. (14) to the ␤Ј-70 complex ( Table 2). In agreement with earlier data, ␤Ј induced a very significant, 90-fold, increase in the interaction with the lacUV5 Ϫ18/ϩ1 oligo. On the other hand, K d values for ␤Ј-70 complex interaction with Ϫ12/ϩ2, Ϫ12/Ϫ3, and Ϫ12/Ϫ6 oligos were comparable with those obtained with free , with the strongest stimulation (for Ϫ2/ϩ2 oligo) being less than 10-fold. The result suggests that there exist favorable interactions between the ␤Ј-70 complex and the upstream segment of the lacUV5 Ϫ18/ϩ1 oligo.
The ␤Ј-70 complex binding to three fork junction probes containing various upstream fragments was also studied. The three probes (Fig. 5A) were designed to reveal if there are any specific interactions between ␤Ј-70 and the Ϫ35 consensus element and the TG motif of extended Ϫ10 promoter element. Comparison of data in Fig. 5B and Table 2 shows that ␤Ј-70 binds fork junctions much more effectively than the Ϫ12/Ϫ3 oligonucleotide, pointing toward the existence of strong favorable interactions between ␤Ј-70 and upstream double-stranded DNA. The highest affinity of the Ϫ35 consensus; TG probe demonstrates the ability of ␤Ј-70 to recognize the extended Ϫ10 element. On the other hand, a small difference in the binding to the Ϫ35 cpnsensus and Ϫ35 mutant probes indicates that the ␤Ј-70 complex is unable to recognize the Ϫ35 element, at least in the context of a fork junction probe.
Interactions of Free 70 Lacking Region 1 and the Corresponding RNAP Holoenzyme with Model Promoter Templates-The N-terminal conserved region 1 of 70 is thought to modulate RNAP holoenzyme interactions with DNA. Region 1.1 is thought to occupy an RNAP trough where double-stranded DNA downstream of the transcription initiation start point is bound in the open promoter complex (17,36). We prepared protein beacons based on 70 truncated at position 104 ((104 -613, 211Cys-TMR) lacks the entire region 1.1 and part of region 1.2) to determine the contribution of conserved region 1 to the binding of oligo and fork junction promoter fragments. The mutant bound oligos with avidity similar to that of the wild-type 70 (Fig. 6 and Table 4). However, in sharp contrast with the data obtained with RNAP containing full-size 70 beacons (Fig. 3), (104 -613, 211Cys-TMR) 70 holo-RNAP bound the Ϫ12/Ϫ6, Ϫ12/Ϫ3, and Ϫ12/ϩ2 oligos with similar affinities that were nearly indistinguishable from free (104 -613, 211Cys-TMR) 70 binding affinities ( Fig. 6 and Table 4). A similar finding was reported by Zenkin et al. (20). The observations thus seem to suggest that 70 region 1 is involved, directly or indirectly, in favorable interactions  We also measured affinity of the mutant holo-RNAP beacon to (Ϫ38/Ϫ7)(Ϫ38/Ϫ12)Ϫ35mut and (Ϫ38/Ϫ3)(Ϫ38/ Ϫ12)Ϫ35mut fork junction probes (shown in Fig. 4) as well as to upstream double-stranded DNA probe (Ϫ38 -12)TG (Table 5). The data show that the binding of fork junctions to mutant RNAP is considerably weaker than to wild-type RNAP, in agreement with previous results (20). Compared with the wild-type holoenzyme, the 45-fold deterioration of the (Ϫ38/Ϫ7)(Ϫ38/Ϫ12)-35mut fork junction avidity conferred by the region 1 truncation is noticeably higher than the 4-fold decrease in the affinity to the Ϫ12/Ϫ6 oligo (Tables 2, 4, and 5). This difference may be explained in part by the overall reduction of mutant RNAP affinity to the doublestranded segment of fork junction probes: indeed, (104 -613, 211Cys-TMR) 70 holoenzyme bound the double-stranded (Ϫ38 -12)TG probe 4.7 less efficiently than the wild-type holoenzyme (Table 5).

DISCUSSION
Interactions of holo-RNAP with promoter elements have been extensively analyzed using oligonucleotides and fork junction model templates (8,33,37). Zenkin et al. (20) have recently shown that, in contrast to previous studies, specific interaction between free 70 and the Ϫ10 element oligonucleotides can also be detected at high (Ͼ1.5 M) oligo concentration. In this study, we applied a new fluorometric assay to quantitative investigation of the interaction between free 70 and the Ϫ10 element oligonucleotides. We found that free 70 interacts with the Ϫ12/Ϫ3 segment of non-template promoter strand. No interaction with other promoter segments in the context of oligonucleotides and fork junction probes as well as with full promoter DNA was detected.
Formation of the holoenzyme increases the apparent affinity of oligonucleotides mimicking the non-template strand of the transcription bubble by ϳ300-fold. However, this increase is mainly a consequence of binding to bases outside of the Ϫ10 element. This result is incompatible with a model that envisions a drastic improvement of specific recognition of the Ϫ10 promoter element by upon the holoenzyme formation as the main reason for highly efficient promoter recognition by the RNAP holoenzyme.
Our data on dissecting the interactions between RNAP and the transcription bubble non-template stand in the context of oligonucleotide and fork junction promoter probes indicate that RNAP binding to the Ϫ10 element and to singlestranded segment located immediately downstream is highly energetically favorable, whereas the interactions with DNA further downstream, around the transcription start point, are weak. This result provides a rationale for biochemical data indicating that on several promoters the downstream part of the transcription bubble melts later than the upstream part, which includes the Ϫ10 element (38 -41). On the other hand, a single molecule study (42) and recent work from Record and co-workers (43) suggested that DNA melting and unwinding occurred in a single step during open promoter complex for-  mation. Clearly, additional experiments employing methods with high temporal resolution will be needed to resolve this issue. It is also probable that the melting pathway may be different on different promoters.
A highly conserved part of 70 region 1, region 1.2, interacts with a promoter segment located immediately downstream of the Ϫ10 element (10,20,44). In the protein beacon assay, the presence of the Ϫ6/Ϫ3 single-stranded segment increased the binding affinity of wild-type RNAP holoenzyme 210-fold (Table 3). In contrast, only ϳ12-fold stimulation was obtained when the RNAP mutant lacking region 1.1 and a part of region 1.2 was used ( Table 5). The residual stimulation may be due to the binding of region 1.2 or may be caused by other interactions, possibly involving RNAP core subunits (11).
The deletions of 70 N-terminal fragments containing region 1 are known to relieve the autoinhibition of free 70 binding to double-stranded DNA fragments containing promoter sequences (46). However, in our work, we did not detect improved binding of short oligos to the (104 -613, 211Cys-TMR) 70 . This discrepancy may be explained by increased nonspecific DNA binding to free 70 without region 1, as was indeed observed (46). In contrast, the beacon assay selectively reports on specific 70 interactions with the Ϫ10 promoter element, which appear to be independent of region 1.
In contrast to free 70 , the ␤Ј-70 complex has noticeable affinity to fork junction probes. As seen from Fig. 5, the TG segment of the extended Ϫ10 element improves binding of a fork junction probe to ␤Ј-70 ϳ9-fold. Thus, the ␤Ј interaction with 70 relieves the autoinhibition effect, which prevents the TG motif interaction with 70 region 3.0 in the context of free 70 . The recognition of the TG motif by a minimal RNAP-melting fragment assembled from the N terminus of the ␤Ј subunit (amino acids 1-314) and amino acids 94 -507 of the 70 subunit was previously observed (45). However the minimal melting fragment lacks 70 segments, which can be involved in the autoinhibition of 70 -DNA interactions (46), complicating interpretation of TG motif interactions with the  RNAP minimal melting fragment. The inability of the ␤Ј-70 complex to recognize the Ϫ35 element in the context of the fork junction probes is in agreement with previous structural and biochemical studies showing an important role of the ␤ flap domain in formation of holo-RNAP conformation able to bind the Ϫ35 element in the context of promoter DNA (16,47,48).
In summary, our work shows that the protein beacon method developed here allows one to measure various RNAP promoter interactions spanning a K d range from picomolar to nearly millimolar. The assay can be instrumental in systematic quantitative dissection of fine details of RNAP interactions with promoters.