Distinct Functions of Regions 1.1 and 1.2 of RNA Polymerase σ Subunits from Escherichia coli and Thermus aquaticus in Transcription Initiation*

Background: RNA polymerases (RNAPs) from Thermus aquaticus and Escherichia coli differ in many aspects of transcription initiation. Results: Regions 1.1 and 1.2 of the σ subunit determine instability and cold sensitivity of promoter complexes of T. aquaticus RNAP. Conclusion: Substitutions in σ regions 1.1 and 1.2 modulate RNAP-promoter interactions. Significance: Evolutionary changes in the σ subunit determine functional differences between bacterial RNAPs during transcription initiation. RNA polymerase (RNAP) from thermophilic Thermus aquaticus is characterized by higher temperature of promoter opening, lower promoter complex stability, and higher promoter escape efficiency than RNAP from mesophilic Escherichia coli. We demonstrate that these differences are in part explained by differences in the structures of the N-terminal regions 1.1 and 1.2 of the E. coli σ70 and T. aquaticus σA subunits. In particular, region 1.1 and, to a lesser extent, region 1.2 of the E. coli σ70 subunit determine higher promoter complex stability of E. coli RNAP. On the other hand, nonconserved amino acid substitutions in region 1.2, but not region 1.1, contribute to the differences in promoter opening between E. coli and T. aquaticus RNAPs, likely through affecting the σ subunit contacts with DNA nucleotides downstream of the −10 element. At the same time, substitutions in σ regions 1.1 and 1.2 do not affect promoter escape by E. coli and T. aquaticus RNAPs. Thus, evolutionary substitutions in various regions of the σ subunit modulate different steps of the open promoter complex formation pathway, with regions 1.1 and 1.2 affecting promoter complex stability and region 1.2 involved in DNA melting during initiation.

Recently, the three-dimensional structure of domain 2 of the Taq A subunit in complex with a short DNA oligonucleotide containing the Ϫ10 element was determined (12). In this complex the nontemplate DNA strand forms tight contacts with region 2 (Fig. 1A), incompatible with double-stranded DNA conformation, explaining previously established roles of region 2 in promoter recognition, DNA melting, and stabilization of RNAP-DNA interactions (1,(13)(14)(15)(16)(17). Although the exact position of the nontemplate DNA strand downstream of the Ϫ10 element remains unknown, structural modeling, site-specific cross-linking, and biochemical analyses suggest that this DNA segment may directly contact region 1.2, depending on the DNA sequence context (10,11,18,19). Amino acid substitutions at various positions of region 1.2 in Eco 70 were shown to decrease RNAP activity, inhibit promoter DNA melting, and destabilize the open promoter complex (Fig. 1B) (19,20). In addition to its direct role in DNA recognition, region 1.2 was shown to allosterically modulate the DNA binding activity of region 2 (21).
The DNA binding activity of primary subunits is inhibited in free s, in part because of the presence of a weakly conserved region 1.1 at their N terminus. Removal of this region in Eco 70 increases specific interactions of 70 with promoters (22,23). As revealed by site-specific cross-linking, in Thermotoga maritima A region 1.1 is physically close to the promoter recognition regions 2 and 4 (24). Although the structures of regions 1.1 in Eco and Taq subunits remain unknown, the structure of region 1.1 from T. maritima was solved by NMR showing that it folds into three ␣ helixes and possesses a negative electrostatic potential on its surface (supplemental Fig. S1) (24). The presence of a high number of negatively charged amino acids is also a characteristic feature of region 1.1 in other primary subunits, including Taq and T. thermophilus A subunits that have highly divergent region 1.1 sequences (supplemental Fig. S1). In addition to the proposed functions in preventing interactions of free with DNA, region 1.1 likely plays an important role in holoenzyme assembly and open complex formation (25)(26)(27)(28). Several studied amino acid substitutions and deletions in this region in Eco 70 (supplemental Fig. S1) decreased RNAP activity, impaired open complex formation, and/or decreased the stability of promoter complexes (25,(27)(28)(29).
Although a wealth of biochemical and genetic data on promoter recognition and transcription initiation were accumulated for Eco RNAP, most structural information on the mechanisms of bacterial transcription was obtained for RNAPs from thermophilic Taq and T. thermophilus that are distantly related to Eco. Although all multisubunit RNAPs share a highly conserved architecture, Thermus RNAPs significantly differ from the Eco RNAP in structural details (30 -33) and transcription properties (18, 34 -39). In particular, Taq RNAP has a higher temperature optimum of activity than the Eco RNAP and is inactive at low and moderate temperatures. These differences were shown to be associated with the catalytic properties of Taq core RNAP and with the properties of the Taq A subunit that is unable to induce promoter DNA melting at temperatures below 45°C (36 -38, 40). The cold sensitivity of promoter opening by the Taq A subunit in comparison with the Eco 70 subunit was explained by substitutions of nonconserved amino acids in region 2 and by differences in the structures of the N-terminal parts of these subunits (36 -38). However, individual roles of A regions 1.1 and 1.2 in transcription initiation by Taq RNAP were not investigated.
A characteristic feature of most studied promoter complexes of Eco RNAP is their very high stability in vitro, with half-lives ranging from tens of minutes to hours. In contrast, promoter complexes of Taq RNAP were shown to be intrinsically unstable and to dissociate within seconds (10,36). The structural features of Taq RNAP that can explain the low stability of promoter complexes formed by this RNAP remain unidentified.
In this work we extended analysis of functional differences between Eco and Taq RNAPs in transcription initiation, with particular emphasis on the roles of regions 1.1 and 1.2 of the 70 and A subunits in promoter complex formation. We demonstrated that both regions 1.1 and 1.2 of the A subunit determine the low stability of promoter complexes of Taq RNAP, whereas substitutions in region 1.2 of A contribute to the cold sensitivity of promoter opening by Taq RNAP, suggesting pos-

from various bacteria.
A, the structure of Taq A domain 2 (amino acids 93-271) in complex with the Ϫ10 DNA element (Protein Data Bank code 3UGO) (12). The nonconserved spacer between regions 1.2 and 2.1 is not shown on the structure. The DNA segment containing the Ϫ10 element (TGTACAAT) is shown in black (backbone) and light yellow (bases), and the first (Ϫ12T) and the last (Ϫ7T) thymines of the Ϫ10 element are indicated. Region 1.2 (dark blue) connects to region 1.1 (blue), the position of which remains unknown. Amino acids substituted in A

EXPERIMENTAL PROCEDURES
RNAPs and Promoters-Wild-type Eco and Taq core RNAPs were purified from E. coli BL21(DE3) cells overproducing all four core RNAP subunits from plasmids pVS10 and pET28ABCZ, respectively, as described previously (35,36,41). Genes coding for mosaic Eco and Taq subunits were generated by PCR mutagenesis of wild-type rpoD genes and cloned between NdeI and EcoRI sites into the pET28 plasmid. Wildtype Eco 70 and Taq A subunits and mosaic subunits, all containing His 6 tags at their N termini, were overexpressed in E. coli BL21(DE3) and purified as described in Refs. 34 and 36.
Promoter DNA fragments for in vitro transcription were obtained as follows. The T7A1, T7A1cons, and T7A1_TGcons promoters (positions from Ϫ85 to ϩ53 nucleotides relative to the starting point of transcription) were obtained by PCR from synthetic oligonucleotide templates. The lacUV5 promoter (positions Ϫ59 to ϩ58) was obtained as described in Ref. 21. The P R promoter fragment (positions Ϫ81 to ϩ54) was obtained by PCR from plasmid pIA226. The sequences of all promoters are shown on Fig. 1C.
In Vitro Transcription-Transcription assays were performed in transcription buffer containing 40 mM Tris-HCl, pH 7.9, 40 mM KCl, and 10 mM MgCl 2 . Holoenzyme RNAPs were prepared by incubating core RNAP (final concentration, 100 nM) and either the wild type or mosaic subunits (500 nM) in the transcription buffer for 5 min at 25°C. DNA template was added (10 -30 nM), and the samples were incubated for 3-5 min at desired temperatures (45°C for holoenzymes containing Eco core RNAP and 55°C for holoenzymes containing Taq core RNAP in most experiments). For analysis of promoter complex stabilities on T7A1 and P R promoters, heparin was added to 10 g/ml. Following incubation of the samples for different time intervals at the same temperatures, transcription reactions were initiated by the addition of dinucleotide primer CpA (25 M) and UTP (10 M, with the addition of [␣-32 P]UTP). The reactions were stopped after 1 min by the addition of an equal volume of buffer containing 8 M urea and 20 mM EDTA; the 3-nucleotide RNA products were analyzed by 23% denaturing PAGE followed by phosphorimaging. For analysis of temperature dependence of transcription on the lacUV5 promoter, reactions were performed in the presence of trinucleotide primer ApApU and UTP (with the addition of [␣-32 P]UTP); the samples were transferred to desired temperatures 5 min prior to addition of nucleotide substrates. For analysis of promoter escape, the reactions were performed in transcription buffer containing 100 mM KCl; all four nucleotide substrates were added (100 M of ATP, CTP, and GTP and 10 M of UTP with the addition of [␣-32 P]UTP), either in the absence or in the presence of the CpA primer (25 M). The transcription reactions were stopped after 5 min, and RNA products were separated by 20% denaturing PAGE.
KMnO 4 Footprinting-For the KMnO 4 footprinting experiments, the lacUV5 promoter was labeled at the 3Ј-end of the template DNA strand with the Klenow fragment of DNA polymerase I and [␣-32 P]dATP as described in Refs. 36 and 42.
Holoenzyme RNAPs (100 nM core and 500 nM ) were incubated with the labeled DNA fragment (10 nM) in the transcription buffer for 10 min at either 25 or 45°C, followed by the addition of KMnO 4 to 2 mM. The reaction was stopped after 20 s by the addition of equal volume of solution containing 1 M ␤-mercaptoethanol and 1 M sodium acetate. DNA was ethanolprecipitated, treated with piperidine as described (6), and analyzed on 10% denaturing polyacrylamide gel.
Nontemplate Oligonucleotide Binding and Cross-linking-Apparent dissociation constants (K d ) for the oligonucleotide binding to RNAP holoenzymes were determined by nitrocellulose filtration method (43). 5Ј-End-labeled nontemplate promoter oligonucleotide containing the Ϫ10 element (see Fig.  5C), taken at fixed 0.03 nM concentration, was mixed with RNAP holoenzyme, taken at varying concentrations (250 nM plus 0.1-100 nM core RNAP), in binding buffer containing 40 mM Tris-HCl, pH 7.9, 10 mM MgCl 2 , and 100 mM NaCl; incubated for 10 min at 25°C; and filtered through 0.45-m nitrocellulose filters (HAWP, Millipore), followed by phosphorimaging. The binding curves were fit to hyperbolic equation where B is a fraction of bound DNA, B max is the maximum binding, and K d is the apparent dissociation constant, using GraFit software (Erithacus Software). RNAP-DNA cross-linking experiments were performed in buffer containing 40 mM Hepes, pH 8.0, 5 mM MgCl 2 , and 100 mM NaCl as described (44). Core RNAP, subunits, and nontemplate oligonucleotide were taken at 50, 300, and 10 nM, respectively. After incubation for 10 min at 25°C, the samples were irradiated for 10 min with a 254-nm UV lamp (4 watts; Spectroline). The DNA-protein complexes were separated by 5% SDS-PAGE.

RESULTS
The Instability of Taq RNAP Promoter Complexes Is Determined by the N Terminus of the A Subunit-To determine which component of the Taq RNAP holoenzyme determines the instability of promoter complexes formed by this RNAP in comparison with the Eco RNAP holoenzyme, we compared stabilities of complexes formed on the T7A1 promoter by wildtype Eco and Taq RNAPs and by a hybrid RNAP containing Eco core and Taq A (Eco/Taq). It should be noted that the properties of promoter complexes formed by a reciprocal hybrid holoenzyme containing Taq core and Eco 70 could not be tested because such holoenzyme is inactive (35,36). For each of the three RNAPs, we measured the kinetics of promoter complex dissociation in the presence of heparin ( Fig. 2A). The measurements were performed at 45°C in the case of Eco and hybrid Eco/Taq RNAPs and at 55°C in the case of Taq RNAP. In agreement with published data, we found that promoter complexes of Eco RNAP were stable with half-life exceeding 10 min. In contrast, promoter complexes of Taq RNAP were unstable and almost completely dissociated within 20 s. The hybrid Eco/ Taq holoenzyme also displayed much lower stability of promoter complexes, with a half-life of about 40 s ( Fig. 2A). Importantly, the hybrid holoenzyme was highly active and fully melted promoter DNA around the starting point of transcription under the same conditions (at 45°C), demonstrating that the low promoter complex stability does not result from its JULY Fig. 5). In addition to the heparin challenge experiments, we analyzed activity of the T7A1 promoter complexes formed by the three RNAPs under various ionic strength conditions. Promoter complexes of Taq RNAP were much more salt-sensitive than promoter complexes of Eco RNAP, and promoter complexes of the hybrid RNAP displayed intermediate salt sensitivity. 4 Thus, we conclude that the low stability of promoter complexes of Taq RNAP holoenzyme is in a large part determined by the properties of the Taq A subunit (but also by the properties of the Taq core enzyme, because the Taq holoenzyme has lower promoter complex stability than the hybrid Eco/Taq holoenzyme).

Functions of Subunit Region 1 in Transcription Initiation
To determine which region of the A subunit is responsible for instability of promoter complexes formed by the hybrid RNAP, we tested several mosaic subunits consisting of various parts of the 70 and A subunits. The exchanged segments of the subunits included the N-terminal part, including conserved region 1 and a nonconserved spacer between regions 1 and 2 (amino acids 1-386 and 1-209 in 70 and A , respectively); conserved region 2 (amino acids 387-455 and 210 -278 in 70 and A , respectively); and the C-terminal part, including conserved regions 3 and 4 (amino acids 456 -613 and 279 -438 in 70 and A , respectively). In total, six mosaic subunits with all possible combinations of these segments (ETE, EET, ETT, TET, TTE, and TEE) were studied (Fig. 2B). All of the mosaic subunits were shown to form active holoenzymes with Eco core RNAP (38). 5 It was found that RNAP holoenzymes that contained the subunits with the N-terminal part from 70 (ETE, EET, and ETT) formed stable promoter complexes, whereas holoenzymes that contained the subunits with the N-terminal part from A formed unstable complexes when challenged with heparin (Fig. 2C). Importantly, the promoter complex stabilities of RNAPs containing mosaic s did not correlate with their abilities to form the open promoter complex at low temperatures (20°C) (Fig. 2B). In particular, the holoenzyme containing the ETE subunit displayed cold sensitivity of promoter opening (38) but formed stable promoter complexes.

Regions 1.1 and 1.2 Together determine the Differences in Promoter Complex Stabilities between Eco and Taq RNAPs-
To more precisely locate the region(s) in the N-terminal part of the subunit that can affect the promoter complex stability in Eco and Taq RNAPs, we designed a second set of mosaic subunits with substitutions of regions 1.1 and 1.2. The mosaic subunits were based either on the 70 or the A sequence and contained replacements of region 1.1 ( teE, amino acid residues 1-93 in 70 replaced with residues 1-91 from A ; etT, residues 1-91 in A replaced with residues 1-93 from 70 ), region 1.2 ( etE, residues 94 -125 in 70 replaced with residues 92-123 from A ; teT, residues 92-123 in A replaced with residues 94 -125 from 70 ), or both ( ttE that contained regions 1.1 and 1.2 from A ) (Fig. 3A). Whereas regions 1.1 in 70 and A significantly differ in their sequences, region 1.2 is highly conserved and contains only 14 substitutions in A in comparison with 70 (Fig. 1B). Thus, to reveal possible effects of individual substitutions in region 1.2, we obtained a 70 mutant that contained two A -specific substitutions, M102L and R103H ( MR-LH), that changed amino acids likely involved in contacts with the nontemplate DNA strand downstream of the Ϫ10 element (see Introduction and Fig. 1).
Analysis of the T7A1 promoter complex stabilities of RNAPs containing Eco core RNAP and 70 -based mosaic subunits 4 N. Miropolskaya and A. Kulbachinskiy, unpublished data. 5 A. Kulbachinskiy, unpublished data. revealed that the substitution of region 1.1 in the teE subunit had the most significant effect on promoter complex stability, measured in the presence of heparin, and decreased it almost to the level of the wild-type A subunit (Fig. 3B, upper panel). Substitution of region 1.2 in etE also decreased promoter complex stability but to a lesser extent. Importantly, the effect of the MR 3 LH substitution in region 1.2 was comparable with the effect of the substitution of the whole region 1.2. Finally, RNAP containing ttE with substitutions of both regions 1.1 and 1.2 displayed the same promoter complex stability as RNAP containing wild-type A (Fig. 3B). Thus, substitutions in these two regions can fully explain the lower stability of promoter complexes formed by the A -containing RNAP on the T7A1 promoter. We then tested the effects of substitutions in the A -based etT and teT subunits on the promoter complex stability. Substitution of region 1.2 in teT slightly increased the stability of promoter complexes in comparison with A Taq. At the same time, substitution of region 1.1 in etT had a stronger stabilizing effect on promoter complexes (Fig. 3B, lower panel).
To test whether the observed effects are general for various promoters, we repeated the experiment with holoenzymes containing Eco core RNAP and various subunits on the P R promoter that forms more stable complexes with Eco RNAP (Fig. 3C). Promoter complexes formed by RNAP containing wild-type 70 were highly resistant to heparin challenge and did not dissociate within 120 min. In contrast, the dissociation kinetics was much faster in the case of A -containing RNAP (t1 ⁄ 2 ϭ ϳ10 min). The half-life times of promoter complexes formed by holoenzymes containing mosaic subunits were also decreased in comparison with wild-type Eco RNAP, although the effects were less dramatic than in the case of the T7A1 promoter. In particular, the promoter complex half-lives for RNAPs containing teE and etE were ϳ50 and ϳ90 min, respectively (Fig. 3C). Thus, it can be concluded that substitutions of regions 1.1 and 1.2 from the A subunit destabilize complexes formed by Eco RNAP on various promoters. Region 1.1 from 70 Can Stabilize Promoter Complexes Formed by Taq RNAP-To determine whether substitutions of regions 1.1 and 1.2 could also affect the stability of promoter complexes formed by Taq RNAP, we analyzed the dissociation kinetics of promoter complexes formed by RNAPs containing Taq core and subunits etT and teT. In contrast to 70 , these two mosaic subunits were shown to form fully active holoenzymes with Taq core RNAP. In the case of the T7A1 promoter, promoter complexes formed by holoenzymes containing both mosaic subunits were unstable and rapidly dissociated after  of complexes formed by holoenzyme RNAPs containing Eco core RNAP and corresponding subunits on the T7A1 and P R promoters, measured in the presence of heparin (10 g/ml), are shown on the right. B, kinetics of dissociation of the T7A1 promoter complexes formed by Eco, Taq RNAPs, and RNAP holoenzymes containing Eco core RNAP and various subunits. The data for 70 -and A -based subunits are shown on the upper and lower plots, respectively. RNAP activities were measured after incubation of promoter complexes in the presence of heparin for various time intervals. For each time point, the level of heparin-resistant activity relative to the activity measured in the absence of heparin is shown. C, kinetics of dissociation of the P R promoter complexes formed by RNAP holoenzymes containing Eco core RNAP and various subunits. the addition of heparin (Fig. 4). We then repeated the experiment on promoter T7A1_TGcons, a variant of the T7A1 promoter containing three consensus promoter elements (Ϫ10, TG, and Ϫ35 elements; Fig. 1C), to increase the strength of RNAP-promoter interactions. On this promoter, holoenzymes containing A and teT also formed unstable complexes. However, holoenzyme containing etT displayed increased promoter complex stability, and a significant fraction of complexes remained active even after 10 min of incubation with heparin (Fig. 4). Thus, substitution of region 1.1 in A with region 1.1 from 70 can stabilize promoter complexes formed by Taq RNAP.
Cold Sensitivity of Transcription by Hybrid Eco/Taq RNAP-Previously, hybrid RNAP containing Eco core and Taq A was shown to be unable to open promoters at moderate temperatures (20 -25°C), suggesting that the A subunit is responsible for the cold sensitivity of promoter opening by Taq RNAP (see Introduction and Refs. 36 and 38). We compared DNA melting by the wild-type Eco and hybrid Eco/Taq RNAP holoenzymes on the model lacUV5 promoter by the KMnO 4 footprinting and confirmed that, in contrast to the Eco holoenzyme, the hybrid holoenzyme was unable to open the promoter at 25°C (Fig. 5A,  lanes 1 and 3). At the same time, both holoenzymes fully opened the transcription bubble at 45°C (Fig. 5A, lanes 2 and  4). Similarly, the hybrid holoenzyme was inactive in lacUV5promoter-dependent transcription at temperatures below 30°C but synthesized RNA as efficiently as wild-type Eco RNAP at higher temperatures (37-45°C) (Fig. 5B).
To confirm that Taq A is able to bind Eco core RNAP and recognize promoter DNA at low temperatures, we analyzed RNAP interactions with short oligonucleotides corresponding to the nontemplate promoter strand and containing the Ϫ10 element (Fig. 5C). Previously, recognition of such oligonucleotides by holoenzyme RNAP was shown to mimic the recognition of the Ϫ10 element in the open promoter complex (44 -46). We found that both wild-type Eco and hybrid Eco/Taq holoenzymes bound the nontemplate oligonucleotide with high affinity, with apparent K d values of 3.1 Ϯ 1.6 and 3.2 Ϯ 2.6 nM at 25°C (see "Experimental Procedures" for details on K d measurements). Furthermore, both RNAPs formed highly efficient cross-links between the corresponding subunit and the nontemplate oligonucleotide upon UV irradiation (Fig. 5C,  lanes 3 and 5). Notably, the efficiencies of Taq A -oligonucleo-tide cross-linking were similar in the case of the hybrid Eco/Taq RNAP and the Taq RNAP holoenzyme (compare lanes 4 and 5), suggesting that the Taq A subunit similarly interacts with the Eco and Taq core RNAPs under our experimental conditions. In contrast, no efficient cross-linking was observed in the case of free 70 and A subunits (lanes 1 and 2) and in the complex of Taq core RNAP and the Eco 70 subunit, which do not form an active holoenzyme (lane 6). These results suggest that the observed cold sensitivity of promoter opening by the hybrid Eco/Taq RNAP is not due to defects in the binding of the heterologous A subunit to the Eco core RNAP and/or promoter DNA recognition but likely results from hampered promoter DNA melting at low temperatures.
A -specific Substitutions in Region 1.2 Increase the Temperature of Promoter Opening by Eco RNAP-Previously, it was shown that the cold sensitivity of DNA melting by the Taq A subunit is explained by structural features of region 2 and of the N-terminal part of A , including regions 1.1 and 1.2 (38). In particular, substitutions of either region 2 or the N-terminal part in Eco 70 with corresponding regions from A (in the  mosaic ETE and TEE subunits; Fig. 2B) resulted in cold sensitivity of promoter opening by Eco RNAP (38). We therefore tested whether substitutions of regions 1.1 and 1.2 in the mosaic 70 and A subunits obtained in this work can affect promoter opening by Eco RNAP. We found that holoenzymes containing the etT and teT subunits did not open the lacUV5 promoter at 25°C (lanes 9 -12), an expected result because these s contained region 2 from A Taq, which by itself imposes the cold sensitivity of promoter opening (38). In contrast, the mosaic teE subunit with substitution of region 1.1 was able to induce DNA melting at both 25 and 45°C (lanes 5 and 6). However, the etE subunit with substitution of region 1.2 did not support DNA melting at 25°C, although it opened the promoter at 45°C (lanes 7 and 8). Thus, A -specific substitutions in region 1.2, but not in region 1.1, increase the temperature of promoter opening by Eco RNAP. Importantly, RNAP containing the etE subunit formed more stable promoter complexes than the teE-containing holoenzyme, suggesting that substitutions of regions 1.1 and 1.2 independently affect different promoter complex properties.

Differences between Eco and Taq RNAPs in Abortive Synthesis and Promoter Escape Are Partially Determined by Regions 2-4-Changes in stabilities of promoter complexes of Eco
RNAP were shown to significantly affect the efficiencies of abortive synthesis and promoter escape (47). Because Taq RNAP forms much less stable promoter complexes than the Eco RNAP, one could expect that it should also differ from Eco RNAP in the promoter escape efficiency. We found that this indeed was the case. To analyze promoter escape, we performed transcription on the T7A1 promoter and its two consensus variants, T7A1cons (containing Ϫ10 and Ϫ35 consensus elements) and T7A1_TGcons (containing Ϫ10, TG and Ϫ35 elements) (Fig. 1C) that are expected to form strong -mediated contacts with RNAP. In the case of Eco RNAP, large amounts of abortive RNAs of various lengths (up to 16 nucleotides) were synthesized during transcription initiation on the consensus promoters (Fig. 6A) (6, 41). Furthermore, the efficiency of the full-length RNA synthesis was significantly decreased in the case of the T7A1_TGcons promoter. In contrast, Taq RNAP synthesized much lower amounts of abortive RNAs, although the level of abortive synthesis was increased on the consensus promoters (Fig. 6A). Taq RNAP was also able to efficiently synthesize the full-length RNA on all three promoter variants.
To determine whether the increased efficiency of promoter escape by Taq RNAP can be explained by the properties of the A subunit, we analyzed transcription by the hybrid Eco/Taq RNAP. In the case of this RNAP, the amounts of abortive RNAs synthesized during initiation were significantly decreased, and the efficiency of promoter escape was increased in comparison with the 70 RNAP holoenzyme (Fig. 6, B and C). Thus, the A subunit promotes more efficient escape to elongation. At the same time, the hybrid RNAP was still less efficient in promoter escape than the Taq RNAP holoenzyme, demonstrating that the core enzyme also contributes to the higher promoter escape efficiency displayed by Taq RNAP (Fig. 6, compare A and B).
We then tested whether regions 1.1 and 1.2 of the 70 and A subunits contribute to the differences in promoter escape between Eco and Taq RNAPs. It was found that RNAP holoenzymes containing Eco core RNAP and teE or etE, bearing regions 1.1 or 1.2 from Taq, were characterized by the same promoter escape efficiencies as RNAP containing wild-type 70 (Fig. 6, B and C). In contrast, RNAP containing the ETT subunit with regions 2-4 taken from the Taq A subunit behaved similarly to the A -containing RNAP holoenzyme. RNAPs containing subunits with individual substitutions of region 2 ( ETE) or regions 3 and 4 ( EET) displayed intermediate promoter escape efficiencies (Fig. 6C). Thus, substitutions of these regions in Eco 70 with the corresponding regions from A facilitate promoter escape by RNAP.

DISCUSSION
While sharing a conserved transcription mechanism, RNAPs from different bacteria may significantly differ in transcription properties, as a result of phylogenetic divergence or adaptation to various life conditions, as in the case of mesophilic and thermophilic bacteria. The differences between mesophilic Eco and thermophilic Taq RNAPs affect various steps of transcription, from promoter recognition and open complex formation to RNA elongation and termination (34, 36 -40). In particular, at

Functions of Subunit Region 1 in Transcription Initiation
the transcription initiation step Taq RNAP displays cold sensitivity of promoter opening, lower promoter complex stability and higher promoter escape efficiency in comparison with Eco RNAP. In this work, we focused on analysis of the roles of the Eco 70 and Taq A subunits in defining specific differences between the Eco and Taq RNAPs during initiation. Below, we discuss possible impact of evolutionary variations in the structure of various regions of the subunit on different steps of the open complex formation by Eco and Taq RNAPs.
Formation of the open promoter complex by Eco RNAP was previously shown to proceed via at least three intermediates: RP c , the closed promoter complex with fully double-stranded DNA; intermediate complex I 1 , in which the downstream DNA duplex is partially bent and placed inside the DNA binding cleft of RNAP; and complex I 2 , containing the open transcription bubble at the starting point of transcription (reviewed in Ref 48). Further isomerization of the unstable I 2 complex into the stable open promoter complex is accompanied by formation of tight contacts of RNAP with the downstream DNA duplex (49 -51). Although forming highly stable complexes on most promoters, Eco RNAP was found to form unstable complexes on stringent-response promoters, such as the ribosomal rrnB P1 promoter (see Ref. 11 and references therein). The rrnB P1 promoter complexes were shown to have a shortened downstream DNA footprint (52) and to display high efficiency of promoter escape (53). It was therefore proposed that these complexes may be trapped at the I 2 step of the open complex formation (49). The properties of complexes formed by Taq RNAP on most promoters are remarkably similar to the properties of the rrnB P1 promoter complexes. Furthermore, Taq RNAP also has a shortened downstream footprint on promoter DNA (54). We therefore speculate that the Taq RNAP promoter complexes may also correspond to the unstable I 2 intermediate previously described for Eco RNAP. However, a detailed kinetic analysis is needed to establish the exact open complex formation pathway by Taq RNAP.
We demonstrated that the instability of promoter complexes of Taq RNAP in comparison with Eco RNAP is determined by both core RNAP and the A subunit. The core-dependent variations in promoter complex stability may be probably explained by the differences in RNAP contacts with downstream DNA. In particular, Taq core RNAP lacks the SI3 domain that was previously hypothesized to form stabilizing contacts with the downstream DNA duplex in promoter complexes of Eco RNAP (49 -51, 55). The -dependent variations in promoter-complex stability were shown to be mainly determined by region 1.1 and, to a lesser extent, region 1.2. Importantly, substitutions of regions 1.1 and 1.2 in the Eco 70 and Taq A subunits affected promoter complex stabilities on both the T7A1 and P R promoters, which are characterized by different heparin sensitivities and may differ in the structures of intermediates formed by RNAP holoenzyme during promoter opening (49,56).
Previously, region 1.1 was proposed to play an important role in the open promoter complex formation, illuminated by deleterious effects of several studied mutations in region 1.1 in the 70 subunit on transcription by Eco RNAP (25,(27)(28)(29). Based on kinetic analysis and FRET measurements, it was hypothesized that region 1.1 binds within the downstream DNA bind-ing cleft in the free Eco RNAP holoenzyme, likely remains bound inside the cleft in the I 1 and I 2 complexes, and is ejected from the cleft upon formation of the tight downstream RNAP-DNA contacts in the open promoter complex (49,50,57,58). Thus, species-specific differences in the structures of region 1.1 in Eco and Taq RNAPs may affect its interactions with core RNAP and downstream DNA in intermediate and open promoter complexes, resulting in changes of their relative stabilities and shifting the equilibrium between them.
Nonconserved amino acids in region 1.2 also contribute to the lower promoter complex stability of Taq RNAP. Remarkably, substitution of just two amino acids in Eco 70 , M102L and R103H, located in the central part of the first of the two ␣ helixes comprising region 1.2 had the same effect on promoter complex stability as substitution of the whole region 1.2. Thus, these two amino acids likely make the main contribution to the observed differences in promoter complex stabilities determined by region 1.2. Previously, alanine substitutions of residues Met-100 through Met-105 in Eco 70 (corresponding to residues Gln-98 -Ile-103 in A ; Fig. 1) were shown to destabilize promoter complexes of Eco RNAP, likely by disrupting RNAP contacts with the discriminator region located downstream of the Ϫ10 element ( Fig. 1B and Ref. 19). In particular, residue Met-102 of Eco 70 (corresponding to Leu-100 in Taq A ; Fig. 1A) was proposed to directly interact with DNA two nucleotides downstream of the Ϫ10 element and its alanine substitution decreased site-specific cross-linking of region 1.2 with DNA (19). Similarly, the instability of the rrnB P1 promoter complexes was proposed to be in part explained by the absence of favorable interactions of region 1.2 with the discriminator region at this promoter (11). Thus, nonconserved substitutions in region 1.2 in Taq A may affect the open complex stability through weakening the -DNA contacts. Importantly, however, in contrast to the previously studied alanine substitutions in 70 , A -specific substitutions do not disrupt interactions of region 1.2 with DNA, because A region 1.2 was shown to specifically recognize the GGGA element downstream of the Ϫ10 element in promoter complexes of Taq RNAP (10,18).
In addition to their effects on promoter complex stability, substitutions of nonconserved amino acids in region 1.2 of the 70 subunit with corresponding residues from the A subunit resulted in cold sensitivity of promoter opening by Eco RNAP, suggesting that region 1.2 is directly involved in DNA melting during initiation. In support of this, mutations in 70 region 1.2 were previously shown to impair promoter opening by Eco RNAP holoenzyme (20). Substitutions of nonconserved amino acids located in the first ␣-helix of region 1.2, including the M102L and R103H (Fig. 1A), can likely directly affect interactions of the subunit with the nontemplate promoter strand and hinder DNA melting at low temperatures. Thus, both the cold sensitivity and the instability of promoter complexes may result from the loss of favorable contacts of region 1.2 with DNA downstream of the Ϫ10 element. In addition, substitutions of other nonconserved residues of region 1.2, not involved in direct contacts with DNA (in particular, in the second ␣ helix of region 1.2), may indirectly affect DNA melting, either by changing -core interactions or by affecting the conformation of domain 2. In support of this, substitutions of conserved residues in this part of region 1.2 in 70 (shown in Fig. 1B above the 70 sequence) also decreased RNAP activity and inhibited open complex formation by Eco RNAP holoenzyme (20).
Previous studies demonstrated that the main role in DNA melting during open complex formation is played by region 2 of the subunit, with conserved aromatic and positively charged amino acids from this region directly involved in interactions with the Ϫ10 promoter element (Fig. 1A) (12)(13)(14)(15)(16)(17)59). Substitutions of nonconserved amino acid residues in region 2 were demonstrated to modulate the temperature of promoter opening and to be in part responsible for the cold sensitivity of DNA melting by Taq RNAP in comparison with Eco RNAP (38). Our results suggest that nonconserved amino acid substitutions in regions 1.2 and 2 can together determine the natural variations in promoter DNA melting in various bacteria.
The instability of promoter complexes formed by Taq RNAP is paralleled by a higher promoter escape efficiency displayed by this RNAP in comparison with Eco RNAP. We propose that these differences in part depend on the properties of the Taq A subunit that was shown to stimulate promoter escape in comparison with the 70 subunit when combined with Eco core RNAP. However, the higher promoter escape efficiency displayed by the A -containing RNAP does not depend on regions 1.1 or 1.2 but is apparently explained by structural features of the C-terminal part of the subunit, including regions 2, 3, and 4. In particular, nonconserved amino acid substitutions in region 2 may likely affect promoter escape through changes in interactions of the subunit with the Ϫ10 promoter element and/or with core RNAP (38), whereas substitutions in regions 3 and 4 may change contacts with the growing RNA transcript during initiation (5,6).
Although analysis of hybrid RNAPs utilized in this work do have some caveats, several lines of evidence support the validity of this approach for comparison of functional properties of subunits from different bacteria. First, the levels of promoterdependent activities at the temperature optima were similar for the wild-type Eco and hybrid RNAPs containing Taq A and mosaic subunits. Second, Taq A in complex with Eco core RNAP efficiently recognized the Ϫ10 promoter element in the nontemplate promoter strand, suggesting that the subunit adopts a proper conformation for DNA binding. Third, many properties of the hybrid RNAPs expectedly reproduced the properties of Taq holoenzyme RNAP. Finally, individual substitutions of regions 1.1 and 1.2 replaced relatively small parts of the subunit and affected a subset of promoter complex properties, thus implying specific functions for these regions in transcription initiation.
In conclusion, our results suggest that regions 1.1 and 1.2 of the Eco 70 and Taq A subunits modulate conformational transitions of RNAP during open complex formation and determine significant differences between the Eco and Taq RNAPs in transcription initiation. Although the functional importance of these differences for the expression of bacterial genes remains to be established, it can be proposed that the observed evolutionary variations in the properties of the subunit may have an important role in transcription regulation.