Conformational Changes of Escherichia coli RNA Polymerase ς70 Factor Induced by Binding to the Core Enzyme

Mutants of RNA polymerase sigma70 subunit from Escherichia coli with unique cysteine residues engineered into conserved region 1 (autoinhibition domain of sigma70), region 2.4 (-10 DNA element binding domain), region 4.2 (-35 DNA element binding domain), and a nonconserved region between regions 1 and 2 were prepared. The chemical reactivity of the cysteine at each position was determined for free sigma70 and sigma70 in complex with the core polymerase and was used as a measure of a conformational response of a particular region of the protein to an interaction with the core polymerase. Both increases and decreases in cysteine reactivity were observed in the presence of core polymerase at several positions in sigma70, providing direct physical evidence for modulation of sigma70 conformation by the core enzyme. Binding of the core polymerase resulted in increased solvent exposure of DNA binding domains of sigma70 and in more complex changes in the autoinhibition domain (region 1). Similar conformational changes in sigma70 were detected using fluorescence probes covalently attached to cysteine residues engineered into sigma70. Thus, the results obtained provided physical evidence supporting a model in which core enzyme allosterically regulates DNA binding activity of sigma70 by "unmasking" its DNA binding domains.

Mutants of RNA polymerase 70 subunit from Escherichia coli with unique cysteine residues engineered into conserved region 1 (autoinhibition domain of 70 ), region 2.4 (؊10 DNA element binding domain), region 4.2 (؊35 DNA element binding domain), and a nonconserved region between regions 1 and 2 were prepared. The chemical reactivity of the cysteine at each position was determined for free 70 and 70 in complex with the core polymerase and was used as a measure of a conformational response of a particular region of the protein to an interaction with the core polymerase. Both increases and decreases in cysteine reactivity were observed in the presence of core polymerase at several positions in 70 , providing direct physical evidence for modulation of 70 conformation by the core enzyme. Binding of the core polymerase resulted in increased solvent exposure of DNA binding domains of 70 and in more complex changes in the autoinhibition domain (region 1). Similar conformational changes in 70 were detected using fluorescence probes covalently attached to cysteine residues engineered into 70 . Thus, the results obtained provided physical evidence supporting a model in which core enzyme allosterically regulates DNA binding activity of 70 by "unmasking" its DNA binding domains.
Escherichia coli RNA polymerase (RNAP) 1 is a multisubunit enzyme that exists as core enzyme (␣ 2 ␤␤Ј) or holoenzyme (␣ 2 ␤␤Ј) (1). Transcription initiation begins with the specific binding of the holoenzyme to promoter DNA. Although core RNAP has the enzymatic activity necessary for transcription, it lacks the ability to specifically bind promoter DNA (1). Therefore, it was suggested that the function of subunit was to confer specific promoter recognition to RNAP (2). There is a variety of E. coli transcription factors that are expressed in response to different growth conditions and environmental stresses and that regulate the expression of genes accordingly (3). The primary 70 factor is responsible for expression of most genes in E. coli (4) and is the focus of these studies.
The role of 70 as the specificity subunit was confirmed by genetic studies that identified two conserved regions of the protein, regions 2.4 and 4.2, as likely candidates for specific protein-DNA interactions with Ϫ10 and Ϫ35 promoter DNA sequences, respectively (5)(6)(7). However, physical interaction between promoter DNA and free 70 could not be shown (8). This paradox was resolved by the finding that the N-terminal sequences in 70 inhibit specific 70 -promoter DNA interactions (9,10). In these studies, it was shown that the glutathione S-transferase fusion proteins containing fragments of lacking the N-terminal sequences (corresponding to conserved region 1) were able to specifically bind DNA fragments containing Ϫ10 and/or Ϫ35 sequences. In contrast, the glutathione S-transferase fusion proteins containing full-length 70 or glutathione S-transferase fusion proteins containing deletion mutants of in which only a part of conserved region 1 was removed did not bind promoter DNA. Further extensions of these studies showed that polypeptide fragments containing region 1 of 70 , when used in trans, could inhibit specific DNA binding of polypeptide fragments containing region 4 of 70 , whereas the activity of fragments containing region 2 was unaffected (10). Based on these observations, a model of 70 -DNA binding modulation by core enzyme was proposed (10). In this model, in free 70 , the N-terminal region 1.1 occludes region 4, preventing its specific protein-DNA interaction with the Ϫ35 region of promoter DNA. It was further suggested that binding of 70 to core RNAP induced an allosteric conformational change in 70 , such that the N-terminal portion of the protein shifted to expose region 4, which could now contact bases in the Ϫ35 region of promoter DNA. Additionally, it was suggested that DNA binding activity of region 2 was also regulated by N-terminal sequences, but most likely not by a simple occlusion mechanism because region 2 DNA binding activity was inhibited by region 1 only in cis, not in trans (10). However, an interaction between residues from region 1 and region 2 has been proposed (11). The self-inhibition of DNA binding activity of subunits may be a common mechanism for other factors evolutionary related to 70 (10).
In this report, we directly test the model in which 70 promoter DNA binding activity is regulated by the core RNAP through a core binding-induced conformational changes in 70 affecting DNA binding domains of the protein (regions 2.4 and 4.2). In our studies we used full-length 70 protein. Our approach was to engineer, using site-directed mutagenesis, cysteine residues at specific locations in regions 1, 2.4, and 4.2. The chemical reactivity of these residues was then used as an indicator of conformational change in both the free 70 and 70 in the holoenzyme. The results obtained showed that core polymerase induced conformational changes in 70 that affected both DNA binding domains and the auto-inhibition domain of the protein.
Cy5 monosuccinimidyl ester (13) was purchased from Amersham Pharmacia Biotech. The succinimidyl ester of Cy5 was converted to maleimide by reaction with the excess of ethylenediamine followed by reaction with maleimidylpropionic acid succinimidyl ester (Sigma). 5 mg of succinimidyl ester of Cy5 was dissolved in 250 l of N,N-dimethylformamide, and 60 l of 1 M ethylenediamine dissolved in water (pH 7.0) was added. The mixture was incubated for 1.5 h at room temperature, and another 25 l of 1 M ethylenediamine was added. After additional 1 h incubation at room temperature, the mixture was diluted to ϳ6 ml with 25 mM triethylammonium acetate buffer, pH 7.0 (Buffer A), containing 2% acetonitrile. The mixture was loaded onto a 10-ml Source 15RPC reverse phase FPLC column (Amersham Pharmacia Biotech), and 100 ml of 0 -50% Buffer B gradient (Buffer B: Buffer A ϩ 95% acetonitrile) was applied at 3 ml/min. Fractions eluting at ϳ26% Buffer B containing amine derivative of CY5 were collected and lyophilized. Dried fractions were dissolved in 500 l of N,N-dimethylformamide and 10 mg of maleimidylpropionic acid succinimidyl ester dissolved in 250 l of N,N-dimethylformamide was added. The mixture was incubated for 1 h at room temperature, was diluted to ϳ8 ml with Buffer A, and was run on a FPLC reverse phase column as described above. Fractions eluting at ϳ27% Buffer B containing the maleimide derivative of CY5 were collected and lyophilized. CY5 maleimide was stored dry at Ϫ70°C until used.
E. coli K12 cell paste was obtained from University of Alabama Fermentation Facility. All other reagents were of the highest purity commercially available.
Site-directed Mutagenesis-All mutants were generated using the CLONTECH Transformer Site-Directed Mutagenesis kit (Palo Alto, CA) according to the manufacturer's instructions with the exception that single-stranded DNA, isolated using phage M13K07 with 100 g/ml ampicillin and 25 g/ml kanamycin selection, was used as the starting material. All sequencing primers and mutagenic oligonucleotides were purchased from Midland Certified Reagent Co. (Midland, TX) or from Ransom Hill Bioscience, Inc. (Ramona, CA). The plasmid containing the rpoD gene encoding 70 under the control of T7 polymerase promoter (pGEMD) was a gift from Dr. A. Ishihama (National Institute of Genetics, Mishima, Japan). The identity of all mutants was confirmed using dideoxy DNA sequencing method (14).
Protein Expression and Purification-Mutant constructs were introduced into the BL21 (DE3) strain (Novagen, Madison, WI). Proteins were expressed and purified as described previously (15). Freshly prepared 1 mM DTT was added to all steps of purification. Concentrations of the 70 proteins were estimated using calculated molar extinction coefficients of 39,040 M Ϫ1 cm Ϫ1 at 278 nm for each single cysteine 70 mutant (16).
Core RNAP was purified from E. coli K12 cells using the method of Burgess and Jendrisak (17) utilizing chromatography on DNA-cellulose, Sephacryl 300 HR, and Bio-Rex 70 anion exchange column (Bio-Rad). Core RNAP was further purified on a 1-ml MonoQ HR 5/5 column (Amersham Pharmacia Biotech) following the method of Hager et al. (18). Peak fractions containing core protein were pooled and concentrated using Microcon 30 microconcentrators (Amicon Inc., Beverly, MA). This additional purification step utilizing chromatography on a 1-ml MonoQ HR 5/5 was necessary to obtain core RNAP preparations free from impurities of wild type (wt) 70 or other proteins of similar size detectable after reaction of core RNAP with Cy5 maleimide or [ 3 H]-Nethylmaleimide. The concentration of core RNAP was estimated spectroscopically by measuring the absorbance at 280 nm and using an extinction coefficient of 0.55 ml/mg (2).
RNA Polymerase Activity Measurements-The activity of 70 mutants was determined after reconstitution with an excess of purified core RNAP using a run-off transcription assay (19), with the fluorescent base analog UTP-␥-ANS (20) replacing the radioactive UTP as described previously (15).
Reactivity of Cysteine Residues in Free 70 -The reactivity (solvent accessibility) of cysteine residue in each single cysteine mutant of 70 was determined by measuring the rate of incorporation of Cy5 maleimide (see Fig. 2A) to 70 mutants. Mutant proteins stored in the storage buffer with 1 mM DTT were run on an FPLC Superdex 200 column to remove DTT and protein aggregates formed upon storage. Typically, 100 l of 50 -150 nM 70 mutants in 50 mM MOPS (pH 7.5), 0.25 M NaCl, 0.1 mM EDTA, 25% glycerol were added to an Eppendorf tube containing 0.5 nmol of dry Cy5 maleimide to start the reaction. The concentration of Cy5 maleimide in the reaction mixture was thus 5 M. At different times, 10-l samples of the reaction mixture were withdrawn, and the reaction was stopped by the addition 5 l of SDS-PAGE sample buffer (36% glycerol, 12% SDS, 150 mM Tris, 6% ␤-mercaptoethanol, 0.03% bromphenol blue). Samples were loaded on 10% SDS-PAGE mini-gels and were run until the dye front (and the excess of unreacted Cy5 maleimide) ran out of the gel.
Wet gels were imaged with STORM (Molecular Dynamics, Sunnyvale, CA) using red fluorescence mode, and the amount of Cy5 incorporated to 70 was determined from digital images of the gels using ImageQuant (Molecular Dynamics). The pseudo-first order rate constants for Cy5 maleimide incorporation to 70 were determined by nonlinear regression using the SCIENTIST program (Micromath, Salt Lake City, UT).
The Effect of Core Polymerase on Reactivity of Cysteine Residues in 70 -Single cysteine mutants of 70 purified on Superdex 200 column were diluted to 50 nM in 50 mM MOPS (pH 7.5), 0.25 M NaCl, 0.1 mM EDTA, 25% glycerol. To prepare holoenzyme samples, a 1.25 molar excess of the core RNA polymerase was added. The reaction with Cy5 maleimide was started by the addition of 20 l of 70 or the holoenzyme to an Eppendorf tube containing 0.1 nmol of Cy5 maleimide (5 M Cy5 maleimide in the reaction mixture). The reaction, depending on the 70 mutant used, was allowed to proceed for 30 s to 3 min. The time of the reaction for each mutant was selected based on results of rate of Cy5 maleimide incorporation experiments described above, such that at the selected time, no more than ϳ50% reaction progress was achieved. Thus, both an increase and a decrease in cysteine reactivity in the presence of core RNAP could be recorded. The core RNAP contains 30 cysteine residues, 7 of which were reported to be accessible for chemical modification in the native core RNAP (21). Thus, it can be calculated that at the core RNAP concentration used in the cysteine reactivity measurements, less than 10% of the Cy5 maleimide present in solution could be used up by its incorporation into the core RNAP. Reactions were terminated by addition of SDS-PAGE sample buffer. Reaction mixtures were analyzed on a 10% SDS-PAGE, and the amount of Cy5 incorporated into 70 was determined by scanning wet gels using STORM and by quantifying with ImageQuant as described above. Labeling

Single Cysteine Mutants of 70
In our approach, we used the reactivity of cysteine residues engineered into specific domains of the protein as indicators of a conformational response of these domains to 70 -core RNAP complex formation. The first step of this approach was to prepare single cysteine mutants of 70 using site-directed mutagenesis. wt 70 contains three cysteine residues at positions 132, 291, 295 and no disulfide bonds (22,23). Initially, a 70 mutant ([⌬cys] 70 ) was constructed in which all endogenous cysteine residues were replaced with structurally similar serine residues. The mutant protein was expressed, purified, and found to exhibit ϳ150% activity of the wt 70 in an in vitro transcription assay (Table I). We therefore concluded that a replacement of all 70 cysteine residues with serine residues had no negative effect on the function of the protein, as has also been observed recently by others (24,25). Also, because transcriptional activity of 70 involves numerous unique stereospecific interactions ( 70 -core, 70 -duplex DNA, and 70single-stranded nontemplate DNA), the preservation of transcriptional activity in [⌬cys] 70 suggests that the conformation of the mutant protein was also not significantly affected by the Cys to Ser change. The increased activity of [⌬cys] 70 compared with the wt 70 protein may be due to the resistance of the mutant to inactivation due to chemical modifications of cysteine (cross-linking, binding of heavy metals, etc.).
In the next step, single cysteine residues were introduced into [⌬cys] 70 . Fig. 1 shows the schematic representation of the primary structure of 70 , in which the positions of introduced cysteine residues are indicated. The logic behind selecting particular sites for introducing cysteines into region 2.4 and 4.2 was as follows. In each region, we selected one residue implicated by previous mutagenesis studies to be directly involved in the function of the particular region. Although incorporating cysteine into these positions could potentially affect DNA binding or transcriptional activity due to the replacement of a residue making a direct contact with promoter DNA, we reasoned that even if this would occur, the cysteine would still be able to report a change in conformation at the critical sites in response to binding core RNAP. Additionally, two sites in each region were chosen that were close to these residues but for which no direct interaction with DNA has been proposed so far. We reasoned that we could use these additional single cysteine mutants to monitor conformational changes in region 2 and region 4 without affecting DNA binding activity. Using this logic, mutants representing region 2.  (7), although this residue is located near residue 437, which was suggested to also be involved in recognition of position Ϫ12 of the promoter (5). Mutations at position 442 have not been described previously.  70 . Arginine at position 588 is located within the second helix of the proposed helix-turn-helix motif of region 4.2 and was presumed to contact the Ϫ33 base of promoter DNA (6). Threonine at position 583 is the last residue in the turn of the proposed helix-turn-helix of region 4.2 and is one residue away from Arg 584 , which was suggested to contact position Ϫ31 of promoter DNA (7). Arg 596 is located outside of the second helix of the region 4.2 helix-turn-helix motif. Point mutations at this position, previously constructed by Siegele et al. (7), were found to have no observable effects on promoter recognition.
Information regarding the possible function of specific residues in region 1 is very limited. Our two mutants in this region are [1C] 70 , in which cysteine was inserted between the initiation Met residue and the second residue of 70 , and [A59C] 70 , located within region 1.1. The residue at position 59 was selected because alanine containing no side chain was unlikely to be involved in any critical interactions, and this residue is close to residue 55, which was previously suggested to be involved in interacting with Trp 433 of region 2.4 in free 70 (11,27). Additionally, a mutant at position 366 ([S366C] 70 ) was prepared. This position is in a nonconserved region of 70 (Fig. 1), within a portion of 70 determined to be necessary for efficient core binding (28). We used the reactivity of a cysteine at this position as an indicator of conformation outside conserved regions 1, 2, and 4.
Each of the single cysteine mutants of 70 was expressed and purified to near-homogeneity. One striking observation made was that the mobility in SDS-PAGE of [R588C] 70 was different from all other mutant 70 and wt 70 (not shown). The calculated molecular mass of 70 is 70,263 Da, but it is known to migrate as a band of apparent molecular mass of around 90 kDa (29). Replacement of arginine with cysteine at position 588 produced a protein that migrated in SDS-PAGE as ϳ70-kDa band (not shown). The changes in SDS-PAGE mobility of 70 in response to a single amino acid replacement has been observed previously (11,27). Analysis of [R588C] 70 mutant on Superdex-200 sizing column indicated that this protein was smaller than wt 70 , and Western blot analysis using a C-terminal antibody as a probe suggested that the C terminus of [R588C] 70 was missing (not shown). It is thus possible that [R588C] 70 was proteolytically more unstable resulting in purification of a truncated protein. Therefore, this protein was not used for further studies.
The activity of each single cysteine mutant was compared with the activity of wt 70 using reconstituted RNAP in an in vitro run-off transcription assay (19). All single cysteine mu-  ). This residue was not previously indicated as a DNA contact residue of 70 but is one residue away from residue 437, for which such a role was suggested before (5). Cysteine residues at positions 1, 442, and 596 had no effect on the activity of the protein, whereas cysteines at positions 59, 366, 440, 583, and 588 resulted in a moderate decrease in the activity of the protein (Ͼ50% of [⌬cys] 70 activity remaining). Among the positions showing moderate decrease in the activity were positions 440 and 588, which were previously indicated as direct DNA contact residues (6,7). The decrease in activity, rather than complete inactivation, of mutants at these positions is not surprising because original mutations at position 440 and 588 were identified as altered specificity mutants with no effect on the activity of the wt promoter (6, 7). The overall design of our experiments was to use the rate of incorporation of thiol-specific reagent Cy5 maleimide ( Fig. 2A) to single cysteine mutants of 70 as a measure of reactivity of unique cysteine residues of these mutants. We used Cy5 maleimide because it can be detected in STORM fluoroimager with femtomolar sensitivity, allowing the use of nanomolar protein concentrations in cysteine reactivity experiments. Because 70 can be easily separated from the remaining RNAP subunits on SDS-PAGE, both reactivity of cysteines in free 70 and in core-bound 70 could be measured. The assumption in this experimental design is that the rate of incorporation of maleimide label into 70 band in SDS-PAGE does indeed correspond to the reactivity of a single cysteine residue of a particular mutant (maleimide can also react with low efficiency with histidine and the ␣-amino group of amino acids (30)). Thus, two control experiments were performed to validate this assumption. In the first experiment (Fig. 2B), we showed that under our experimental conditions, maleimide incorporation was entirely due to reaction with cysteines. This conclusion is based upon observation that no maleimide incorporation was observed in the case of [⌬cys] 70 , free or core-bound (Fig. 2B), whereas efficient maleimide incorporation under the same conditions was observed in the case of wt protein (Fig. 2C, lanes  1-3). In the second experiment (Fig. 2C, lanes 4 -6), we showed that the preparation of the core RNAP used to prepare holoenzymes with single cysteine mutants of 70 was free of wt 70 contamination or any other significant impurity that would incorporate maleimide, co-migrate with 70 on a SDS-PAGE, and thus complicate data analysis.

Reactivity of Cysteine Residues in Single Cysteine Mutants of 70
Reactivity (solvent exposure) of cysteine residues engineered to different positions of 70 was probed by studying the kinetics of the reaction of single cysteine mutants of 70 with Cy5 maleimide. Fig. 3A shows an example of SDS-PAGE analysis of a time course of Cy5 maleimide incorporation to [T440C] 70 . Fig. 3B shows results of quantitative analysis of the data from Fig. 3A. The time course of Cy5 incorporation could be described by a simple pseudo-first order kinetics (Fig. 3B). shows a summary of cysteine reactivity data for all single cysteine mutants of 70 . In all single cysteine mutants of 70 , cysteine residues were accessible to modification with Cy5 maleimide, albeit with rates differing by as much as 10-fold. The highest reactivity of cysteine was observed for cysteine at position 596, whereas the lowest reactivity was observed for cysteine at position 442. For a segment of 70 for which the crystal structure is available (26), it was possible to compare cysteine reactivity data with solvent accessibility (Fig. 4, inset) of these residues calculated from x-ray coordinates. To accomplish this, the models of single cysteine mutants of 70 were built using the WHAT IF program 2 and a relative solvent accessibility (solvent accessibility of cysteine in 70 mutant compared with solvent accessibility of completely exposed cysteine residue) of cysteine residues was calculated using WHAT IF. Comparison of the body and the inset of Fig. 4 shows that although some correlation between solvent accessibility and cysteine reactivity at positions 366, 438, 440, and 442 exists, the correlation is not strict. The most striking discrepancy between cysteine reactivity and solvent accessibility data was observed for cysteine at position 438. The reactivity of this cysteine was much higher than expected from solvent accessibility data. One possible explanation of this discrepancy would be that replacing alanine (which has essentially no side chain) at position 438 with cysteine could produce a local perturbation of protein conformation. An alternative explanation could be that the available crystal structure is for a fragment of 70 corresponding to ϳ50% of the full-length protein. The differences between results obtained with full-length protein (cysteine reactivity data) and the fragment (crystal) may simply reflect the absence of some interactions in the fragment of 70 that are possible in the full-length protein.

Conformational Changes of 70 Induced by Core RNAP Binding
Cysteine Reactivity-Cysteine reactivity can be used as a sensitive indicator of conformational changes in proteins (31). The reactivity of a cysteine residue in a protein molecule is determined by its solvent accessibility and its pK a value (32). Both of these properties can be changed when the local conformation of the protein in the vicinity of the cysteine changes. The effect of core RNAP on reactivity of cysteine residues in 70 mutants is presented in Fig. 5, A and B, and summarized Fig. 5B). Generally, three classes of cysteine residues can by identified from inspection of Fig. 5C. The first class consists of residues 438 and 442, the reactivity of which remained unchanged in the presence of the core RNAP. Both of these residues are located in region 2.4 of 70 . The second class consists of residues 1, 440, 583, and 596, the reactivity of which was increased in the presence of core RNAP. These residues are located in regions 1, 2.4, and 4.2, respectively. The third class consists of residues 59 and 366, the reactivity of which was decreased in the presence of core RNAP. These residues are in region 1 and in the nonconserved region of the protein, respectively. Decrease of the reactivity of a given cysteine residue in the presence of core RNAP could be due to a direct steric protection by the core enzyme or could be due to a core-induced conformational change in 70 . However, an increase in cysteine reactivity could be only due to a core-induced conformational change in 70 . Thus, results presented in Fig. 5C, provide a direct evidence that a large scale conformational change in 70 is triggered by an interaction with the core RNAP.
Fluorescence Spectroscopy-Conformational changes in 70 induced by binding to the core enzyme were also studied using fluorescence signal of IANBD covalently attached to several selected single cysteine mutants of 70 (Fig. 6). The fluorescence intensity and/or position of fluorescence emission maximum of this probe is dependent on its microenvironment, making its fluorescence a sensitive indicator of conformational changes in proteins (33). A set of representative cysteine mutants of 70 was chosen for these studies such that IANBD fluorophore was introduced to regions 1, 2.4, and 4.2 and to a nonconserved region of the protein (residue 366). Fig. 6 shows that binding of 70 to the core enzyme resulted in significant alterations of IANBD fluorescence at all positions studied, with the exception of position 442, where the changes were insignificant (Fig. 6C). This result is consistent with cysteine reactivity measurements (Fig. 5), in which no change of reactivity at position 442 was observed upon binding of 70 to the core. At the remaining positions (59, 440, 596, and 366), very significant changes of fluorescence intensity and shifts in position of emission maximum were observed. Both an increase (positions 59 and 440) and a decrease (positions 366 and 596) of fluorescence intensity were observed. Also, both red shifts in the maximum emission position (positions 440, 596, and 366) and a blue shift (position 59) were observed. The red shifts of emission maximum at positions 440 and 596, indicating more polar environment of the probe in the holoenzyme, are consistent with the increased solvent exposure of these residues detected by cysteine reactivity measurements (Fig. 5). The blue shift of the emission at position 59, indicating a less polar environment of the probe in the holoenzyme, is also consistent with a decrease in solvent exposure detected by cysteine reactivity experiments at this position (Fig. 5). However, the red shift observed at position 366 is not consistent with the decreased reactivity of this cysteine in the holoenzyme. Similar results were obtained with acrylodan-labeled 70 mutants (not shown).
Overall, fluorescence data support cysteine reactivity data and provide additional evidence that large scale conformational changes in 70 occur upon binding to the core enzyme and that these changes affect at least regions 1, 2.4, and 4.2.

DISCUSSION
Cysteine residues at several positions in regions 2.4 and 4.2, which contain promoter recognition elements, become more accessible to Cy5 maleimide when 70 binds to the core RNAP. The increased accessibility could be caused by an increased solvent exposure of these cysteines or by a change in the pK a values for cysteine. Irrespective of the exact nature of cysteine reactivity increase, these results clearly indicate that regions 2.4 and 4.2 undergo a major conformational change induced by 70 -core interaction. Because the increase of cysteine reactivity is observed in several locations within regions 2.4 and 4.2, it is unlikely that a change in pK a of these cysteines would be responsible for the observed increased cysteine reactivity in the holoenzyme. Such interpretation would require that core-induced conformational changes in 70 produced very similar changes in pK a of cysteine in different locations of 70 . Therefore, it is more likely that the increased reactivity of cysteines simply reflects an increase in solvent exposure of regions 2.4 and 4.2 induced by core RNAP. This conclusion is further supported by fluorescence experiments, which were consistent with increased solvent exposure of fluorescence probes at several locations in region 2.4 and 4.2 in the presence of core RNAP. Therefore, taken together, these data provide direct experimental evidence that binding of 70 to the core polymerase induces "unmasking" of DNA binding domains of 70 . Such unmasking of DNA binding domains is likely to be important for the ability of 70 to bind promoter DNA and is consistent with the model for regulation of 70 -DNA binding by the core RNAP activity proposed by Dombroski et al. (9,10).
The N-terminal region of 70 (region 1) was previously suggested to be involved in masking the DNA binding domains in the free 70 (9,10). This region was proposed to interact with region 2.4 and/or region 4.2 in the free protein and was proposed not to interact with any domains of the protein in 70 bound to the core RNAP. Thus, this model predicts only an increase in solvent accessibility in region 1 upon binding to the core RNAP. We observed an increase in solvent accessibility of cysteine at position 1, but a decrease of accessibility was observed at position 59. Thus, the changes within region 1 as a result of core RNAP binding must be more complex than described by the model. In particular, decrease of reactivity of cysteine at position 59 suggests that in holoenzyme, a new interdomain interaction involving region 1 (in the vicinity of residue 59) is formed or that upon interaction with core, region 1 undergoes a conformational transition itself.
It is interesting to compare solvent accessibility deduced from our cysteine reactivity data with the presumed function of some of these residues. Previous studies determined that mutations of threonine 440 could compensate for promoter downmutations (7). It was suggested that this residue directly contacts promoter at the Ϫ12 position. The expectation was thus that Thr 440 would be solvent-exposed in the holoenzyme in order to make direct DNA contact. We found this residue to be relatively exposed in free 70 , and its exposure was increased upon interaction with the core RNAP, consistent with a direct DNA contact role proposed for this residue. Position 366 is located within the domain of 70 that is indicated by deletion mutagenesis experiments to be involved in 70 -core interactions (28). The crystal structure of 70 fragment shows this residue to be on the periphery of the hydrophobic face proposed to interact with core RNAP (26). Cysteine at this position is reactive in free 70 , and its accessibility is decreased in the holoenzyme. Fluorescence probe at this position upon core RNAP binding exhibited spectral changes (red shift in fluores-cence emission maximum) characteristic for transfer of the probe to a more polar environment. The simplest interpretation of these observations is that residue 366 is near (but not at) the core binding site of 70 . Formation of 70 -core RNAP complex would reduce (but not eliminate) accessibility of cysteine to modification with Cy5 maleimide. The fluorescence changes suggest that some polar residues of bound core RNAP could be located in the vicinity of residue 366 of 70 . Arginine at position 588 is located in the second helix of the putative helix-turnhelix of region 4.2 and was predicted to contact Ϫ31 and Ϫ33 positions of promoter DNA, respectively (7). We found that replacement of Arg 588 with cysteine resulted in a protein that in comparison to wt 70 had altered electrophoretic mobility and was likely to be more susceptible to degradation but was active in a transcription activity assay. This was a rather unexpected observation because a charged arginine residue should be located on the surface of the protein exposed to a solvent. Replacing such a residue should not affect protein structure significantly. Thus, it is likely that arginine at position 588 in free 70 is involved in some interactions important for maintaining the structure of the protein. One obvious candidate could be an interaction between regions 1 and 4.2.
In summary, we present here a direct evidence for core RNAPinduced large scale change in conformation of 70 resulting in unmasking of DNA binding domains (region 4.2 in particular). The next step now should be an elucidation of the molecular mechanism by which these conformational changes are accomplished.