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Journal of Bacteriology, April 2008, p. 3088-3092, Vol. 190, No. 8
0021-9193/08/$08.00+0     doi:10.1128/JB.00008-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Lineage-Specific Amino Acid Substitutions in Region 2 of the RNA Polymerase {sigma} Subunit Affect the Temperature of Promoter Opening{triangledown} ,{dagger}

N. Barinova,1 E. Zhilina,1 I. Bass,1 V. Nikiforov,1,2 and A. Kulbachinskiy1*

Institute of Molecular Genetics, Kurchatov Sq. 2, Moscow 123182, Russia,1 Public Health Research Institute, 225 Warren Street, Newark, New Jersey 071032

Received 3 January 2008/ Accepted 5 February 2008


   ABSTRACT
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Highly conserved amino acid residues in region 2 of the RNA polymerase {sigma} subunit are known to participate in promoter recognition and opening. We demonstrated that nonconserved residues in this region collectively determine lineage-specific differences in the temperature of promoter opening.


   TEXT
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Promoter opening is a temperature-dependent process that requires conformational changes in both RNA polymerase (RNAP) and DNA (4). In bacteria, promoter recognition and opening are accomplished by the RNAP holoenzyme, which consists of a core enzyme and a specificity factor, the {sigma} subunit. DNA melting is initiated through specific interactions of conserved region 2 of the {sigma} subunit with the –10 promoter element (consensus sequence, TATAAT) (7). Genetic and biochemical analyses have implicated conserved amino acids from different subregions of region 2 both in interactions with core RNAP and in DNA melting. Mutations of conserved residues in subregions 2.1 and 2.2 affected {sigma}-core interactions and defined this part of region 2 as the main core binding site of {sigma} (Fig. 1A) (20). Mutations in a cluster of highly conserved aromatic residues in subregion 2.3 of Escherichia coli {sigma}70 and Bacillus subtilis {sigma}A were shown to lead to severe defects in promoter opening that could be partially suppressed by an increase in the temperature (Fig. 1A) (6, 9, 16). It was therefore proposed that these amino acids may directly initiate DNA melting through hydrophobic interactions with nucleotide bases of the –10 element. Particular attention has been paid to Y430 and W433 (E. coli numbering), which were proposed to interact with a conserved adenine at the second position of the –10 element (position –11A) in the nontemplate DNA strand (reference 18 and references therein). Mutations of other conserved residues in subregions 2.2, 2.3, and 2.4 (including basic amino acids in subregion 2.3) also had dramatic effects on promoter opening (Fig. 1A). These amino acids were proposed to play a role in the recognition and/or proper positioning of promoter DNA (6, 7, 21). At the same time, the detailed mechanism of promoter melting remains unknown due to the absence of information on the high-resolution structure of the open promoter complex.


Figure 1
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  		FIG. 1. Mutations in region 2 of the E. coli {sigma}70 subunit. (A) Alignment of the sequences of region 2 of the major {sigma} subunits from different bacterial species (Eco, E. coli; Taq, T. aquaticus; Dra, D. radiodurans; Rpr, Rickettsia prowazekii; Aae, A. aeolicus; Hpy, Helicobacter pylori; Tma, T. maritima; Mtu, M. tuberculosis; Bsu, B. subtilis). Amino acid numbers are indicated on the left. The positions of {sigma} subregions are indicated at the top (green, subregion 2.1; light blue, subregion 2.2; yellow, subregion 2.3; violet, subregion 2.4). The letters above the alignment indicate previously studied mutations in region 2 of E. coli {sigma}70 (core binding, mutations leading to defects in core RNAP binding [20]; open complex formation, mutations leading to defects in promoter binding and opening [6, 16, 21]). Mutations whose effects on open complex formation can be partially suppressed at an increased temperature are indicated by asterisks. The letters below the alignment indicate mutations in B. subtilis {sigma}A that result in temperature-dependent defects in promoter melting (9). (B) Alignment of sequences of region 2 of {sigma} variants studied in this work. E. coli amino acid positions are indicated above the sequences. Taq and Dra, {sigma} subunits from T. aquaticus and D. radiodurans, respectively; ETE, {sigma}70 subunit containing whole region 2 from T. aquaticus; M1-2, M3, and M4, {sigma}70 variants having changes in subregions 2.1 and 2.2 (plus one substitution in subregion 2.3), subregion 2.3, and subregion 2.4, respectively; M3-4, M1-3, and M1-2;4, {sigma}70 variants with combinations of changes in different subregions of region 2; EBE, {sigma}70 subunit containing region 2 from B. subtilis {sigma}A. Amino acids that are substituted in the mosaic {sigma}s are indicated by a red background; amino acid changes unique to B. subtilis {sigma}A are indicated by a gray background. The abilities of RNAPs containing various {sigma}s to melt promoter DNA at 20°C are indicated on the right. Amino acid changes that likely have the strongest effect on the temperature of promoter opening are indicated by arrows below the sequences.

 
In RNAPs from different species there are certain functional differences in promoter recognition and opening which are believed to result, at least in part, from their different adaptations (1, 3). In particular, RNAPs from thermophilic bacteria have long been known to melt DNA and initiate transcription at higher temperatures than their mesophilic counterparts (12, 13, 15, 17, 24). In accordance with this, we recently demonstrated that RNAP from the thermophilic bacterium Thermus aquaticus opens promoters at temperatures above 37°C but, unlike RNAP from E. coli, is unable to open promoters at lower temperatures (10). Surprisingly, we found that RNAP from the mesophilic organism Deinococcus radiodurans, which is closely related phylogenetically to T. aquaticus, had similar cold sensitivity of promoter opening, implying that this property is not connected to thermophilic adaptation but may result from adaptation to some other, unknown conditions. Furthermore, we demonstrated that the cold sensitivity of promoter opening by these RNAPs is determined by the {sigma} subunit since hybrid holoenzymes consisting of the E. coli core and {sigma}s from either T. aquaticus or D. radiodurans were also unable to open promoters at low temperatures (10). Remarkably, {sigma}A subunits from both T. aquaticus and D. radiodurans are identical to E. coli {sigma}70 at most positions of region 2 that were previously analyzed by mutagenesis and implicated in promoter melting (Fig. 1A). Thus, in the present study we aimed to identify the structural features of {sigma} that account for the observed cold sensitivity of promoter opening by T. aquaticus and D. radiodurans RNAPs.

To identify the {sigma} region(s) responsible for the functional differences between RNAP from E. coli and RNAPs from T. aquaticus and D. radiodurans, we constructed three mosaic {sigma}70 subunits in which different regions were replaced with corresponding parts from T. aquaticus {sigma}A. In the first mosaic ({sigma}ETE) (Fig. 1B), we replaced region 2 (T. aquaticus amino acids 210 to 278 substituted for E. coli amino acids 387 to 455); the other two mosaics contained changes at either the N terminus ({sigma}TEE; T. aquaticus amino acids 1 to 209 substituted for E. coli amino acids 1 to 386) or the C terminus of {sigma}70 ({sigma}EET; T. aquaticus amino acids 279 to 438 substituted for E. coli amino acids 456 to 613). Genes coding for the mosaic {sigma}s were generated by PCR mutagenesis of wild-type rpoD genes of E. coli and T. aquaticus and cloned between NdeI and EcoRI sites into pET28. Wild-type and mosaic {sigma}s were overexpressed in E. coli BL21(DE3) and purified as previously described (10). Control experiments demonstrated that all three mosaic {sigma}s formed active holoenzymes with E. coli core RNAP and directed efficient promoter-dependent RNA synthesis at temperatures greater than 37°C with both linear and supercoiled DNA templates containing either lacUV5 or T7A1 promoters (data not shown). At the same time, neither of the mosaic subunits formed active hybrids with T. aquaticus core RNAP (data not shown). This is consistent with previous reports that E. coli {sigma}70 is unable to activate T. aquaticus core RNAP (10, 11).

The ability of RNAPs containing mosaic {sigma} subunits and the E. coli core to melt promoter DNA was studied by using KMnO4 footprinting of lacUV5 promoter complexes on a linear DNA template at 20 and 45°C as previously described (10). For each RNAP, the ratio of cleavage efficiencies at these temperatures was calculated for position –3 of the nontemplate promoter strand and used to estimate the level of cold sensitivity (Fig. 2). All RNAPs melted DNA around the starting point of transcription at 45°C (Fig. 2, lanes 2, 4, 6, 8, and 10), which was consistent with their ability to initiate RNA synthesis under these conditions. In agreement with previously described data (10), a holoenzyme containing the wild-type {sigma}70 subunit opened the promoter at 20°C, while a holoenzyme containing T. aquaticus {sigma}A did not open the promoter at 20°C (the ratios of the cleavage efficiencies at 20 and 45°C for these RNAPs were 1.11 and 0.07, respectively) (Fig. 2, lanes 1 and 3). Only one of the mosaics ({sigma}EET, containing regions 3 and 4 from T. aquaticus) melted DNA at the low temperature, while the two other mosaics, having changes either in region 2 ({sigma}ETE) or in the N terminus ({sigma}TEE), were deficient in promoter opening (the ratios of the cleavage efficiencies for the mosaics were 1.02, 0.19, and 0.09, respectively) (Fig. 2, lanes 5, 7, and 9). Further analysis of amino acid changes that might account for the cold sensitivity in the case of {sigma}TEE is complicated due to low sequence conservation in the N-terminal part of {sigma} (see below). We therefore focused on analysis of functionally important changes in region 2 in which {sigma}As from T. aquaticus and D. radiodurans where different from E. coli {sigma}70 at only 12 and 10 positions, respectively (Fig. 1B).


Figure 2
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  		FIG. 2. KMnO4 probing of promoter opening by RNAPs containing various {sigma} subunit variants at 20 and 45°C. The lacUV5 promoter was labeled at the 3' end of the nontemplate strand as described previously (10). The concentrations of E. coli core RNAP, {sigma} subunit variants, and promoter DNA in the samples were 50, 500, and 10 nM, respectively. The positions of modified thymine residues in the melted promoter region are indicated on the left. Lane M contained an A+G sequencing marker. The numbers at the bottom indicate the ratio of cleavage efficiencies at 20 and 45°C (20/45) for promoter position –3 (in each case, the data were normalized to the total amount of DNA in the sample). For each RNAP, the experiment was repeated three or four times, and the average value was calculated. Eco, E. coli; Taq, T. aquaticus; 1-2, 3, 4, 3-4, 1-3, and 1-2;4, mutants M1-2, M3, M4, M3-4, M1-3, and M1-2;4, respectively; t, temperature.

 
To identify particular amino acids that could affect the temperature of DNA melting, we constructed two sets of mosaic {sigma} subunits based on {sigma}70 in which individual subregions of region 2 were replaced with homologous sequences from T. aquaticus (Fig. 1B). In the first three mutants, mutants M1-2, M3, and M4, we replaced subregions 2.1 and 2.2 (seven amino acid substitutions, including one substitution in subregion 2.3), subregion 2.3 (three amino acid changes), and subregion 2.4 (two changes), respectively. The mutants in the second set had combinations of changes in subregions 2.3 and 2.4 (mutant M3-4), subregions 2.1, 2.2, and 2.3 (mutant M1-3), and subregions 2.1, 2.2, and 2.4 (mutant M1-2;4). In the M1-3 mutant, we introduced a single amino acid change into the middle of subregion 2.3 (G424R), compared to three changes in T. aquaticus {sigma}A, since only this substitution is present in subregion 2.3 of {sigma}A from D. radiodurans, which is also cold sensitive. All mutant {sigma} subunits were obtained as described above. As expected, all mutants formed active holoenzymes with E. coli core RNAP and promoted efficient promoter melting at 45°C (Fig. 2, lanes 11 to 24, and data not shown). All three mutants in the first set (M1-2, M3, and M4) were able to open the promoter at 20°C (although the M1-2 mutant opened it with lower efficiency), and none of them had the same level of cold sensitivity of promoter opening as the original {sigma}ETE variant (Fig. 2, lanes 11, 13, and 15). Only one of the remaining three mutants, M3-4, melted DNA at 20°C (Fig. 2, lane 17), while the two others (M1-3 and M1-2;4) were unable to open the promoter. Control experiments demonstrated that RNAPs containing mosaic {sigma}s bound the promoter at 20°C with similar efficiencies, indicating that the substitutions in region 2 specifically affected DNA melting but not holoenzyme formation and/or promoter recognition (see Fig. SA1 in the supplemental material).

At many of the nonconserved positions in region 2 at which the residues are different in {sigma} subunits from E. coli and T. aquaticus there are also differences in {sigma}s from other bacteria, including thermophilic species (e.g., Thermotoga maritima and Aquifex aeolicus) and mesophilic species (e.g., Mycobacterium tuberculosis and B. subtilis) (Fig. 1A). To obtain additional information on the role of these differences in promoter melting, we constructed a mosaic {sigma}70 subunit in which region 2 was replaced with the sequence from B. subtilis {sigma}A ({sigma}EBE) (Fig. 1B). We chose the sequence from B. subtilis since (i) promoter melting by B. subtilis RNAP has been extensively studied previously and mutations in {sigma}A subregion 2.3 had effects on DNA melting similar to those of the mutations in {sigma}70 (9), and (ii) in this sequence there is a distinct set of substitutions, some of which are different from and some of which are identical to substitutions in {sigma}As from D. radiodurans and T. aquaticus. KMnO4 probing experiments revealed that RNAP containing {sigma}EBE is able to melt DNA at both 20 and 45°C (Fig. 2, lanes 23 and 24) and, therefore, substitutions in region 2 of B. subtilis {sigma}A do not affect the temperature of promoter opening.

The results of our study demonstrate that the differences in the temperature of promoter opening by RNAPs from E. coli, T. aquaticus, and D. radiodurans are determined by amino acid changes in two different regions of {sigma}, namely, conserved region 2 and the N-terminal part of the subunit. The poorly conserved N-terminal part (including conserved region 1 and a nonconserved linker between regions 1 and 2) differs at too many positions in E. coli and T. aquaticus to allow unequivocal identification of particular amino acids that could affect promoter melting. However, based on available structural and biochemical data, one can speculate that the functionally important changes may lie (i) in negatively charged region 1.1, which binds within the downstream DNA binding channel of the core (14) and affects open complex stability on various promoters (23), and/or (ii) in region 1.2, which has been shown to interact with the discriminator region downstream of the –10 promoter element (5, 8) and to stimulate the DNA binding activity of {sigma} region 2 (25). The differences in the nonconserved linker may also affect promoter melting since this region makes extensive contact with the β' subunit that, in turn, interacts with {sigma} region 2 (see below).

Analysis of substitutions in region 2 of {sigma}s from E. coli, T. aquaticus, and D. radiodurans revealed that no single amino acid residue can account for the differences in promoter opening by the RNAPs; rather, residues in different subregions of region 2 contribute together to control the temperature of DNA melting. Thus, cold sensitivity of promoter opening can be imposed on the {sigma}70 subunit by a combination of changes at nonconserved positions in subregions 2.1 and 2.2 with changes in subregion 2.3 and/or subregion 2.4. This suggests that these substitutions have a concerted effect on promoter opening and coevolved in the Thermus-Deinococcus lineage. In wild-type T. aquaticus {sigma}A, the cold sensitivity caused by changes in region 2 is likely further enhanced by substitutions in the N-terminal part of {sigma}. The cold-sensitive mosaic {sigma}s M1-3 and M1-2;4 contain a total of 10 substitutions in region 2 (Fig. 1B). Four of these substitutions (I388V, N396G, D417E, and S442A) are also found in {sigma}s from B. subtilis and other bacteria (Fig. 1A) and were introduced into {sigma}EBE, which did not exhibit cold sensitivity. The cold sensitivity of the mosaic {sigma}s, therefore, likely resulted from the remaining six substitutions, as shown in Fig. 1B. Based on available biochemical and structural data, we propose the following possible functions for different groups of these substitutions in promoter opening.

(i) Three of four substitutions in subregions 2.1 and 2.2 (Q400S, I410Q, and M413I) are located on a {sigma} surface that interacts with a coiled-coil motif of the β' subunit of core RNAP and cannot directly interact with promoter DNA (Fig. 3) (14, 22). Mutations of amino acids in this {sigma} region (including M413, one of the residues changed in our study) were previously shown to weaken the {sigma}-core interaction (Fig. 1A) (20). These changes therefore likely have an indirect effect on promoter melting, by affecting {sigma} interactions with the core and changing local conformation of subregions 2.3 and 2.4. At the same time, these substitutions did not impair holoenzyme formation (at least under the conditions used in our experiments) since the hybrid holoenzymes efficiently bound promoter DNA at 20°C and melted it at 45°C. Our results for the first time demonstrate that changes at the {sigma}-core interface can specifically affect the DNA melting function of {sigma} region 2 and suggest that nonconserved amino acid substitutions in the β' coiled coil, in combination with changes in {sigma} region 2, may also modulate the temperature of promoter opening by different RNAPs. Thus, analysis of the possible role of substitutions in the β' coiled-coil region of RNAPs from thermophilic and mesophilic bacteria (including the enzymes from T. aquaticus and D. radiodurans that differ from E. coli RNAP at seven and eight positions in this region, respectively) in promoter opening is an important goal of future studies.


Figure 3
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  		FIG. 3. Location of substitutions in {sigma} on the three-dimensional structure of T. thermophilus RNAP holoenzyme (22). {sigma} region 2 (the colors used for subregions of region 2 are the same as those in Fig. 1) and the β' coiled-coil region (dark blue; amino acids 263 to 307 [E. coli numbering]) are shown. Amino acid residues of {sigma} that are different in E. coli and T. aquaticus are indicated by stick models; six amino acid changes that most likely account for the cold sensitivity of promoter opening are light yellow and are labeled. Y430 and W433 are dark yellow. The proposed location of the –10 element in the nontemplate promoter strand (14) is indicated by a dashed line.

 
(ii) Substitution G424R in subregion 2.3 is located in a turn between two {alpha} helixes implicated in interactions with core RNAP and DNA (Fig. 3). It should be noted that this substitution is unique to the Thermus-Deinococcus lineage and is not found in primary {sigma} subunits from other bacteria (Fig. 1A) (V. Nikiforov, unpublished data). Further analysis of the origin of this substitution may therefore provide interesting insights into the evolution of bacterial RNAP. Owing to the location and the character of this mutation (a highly flexible Gly is replaced by Arg), its effect on promoter melting is likely explained by a decrease in conformational flexibility of region 2. Indeed, previous studies demonstrated the importance of local flexibility of this region for open complex formation (2). Remarkably, the effect of the G424R substitution on promoter opening has been observed only in combination with substitutions in subregions 2.1 and 2.2. In agreement with this, it has been reported that a single complementary R247G substitution in T. aquaticus {sigma}A did not affect promoter binding by T. aquaticus RNAP holoenzyme (19). At the same time, this substitution enhanced RNAP interactions with single-stranded DNA, suggesting a role for this amino acid in promoter opening (19).

(iii) Substitution K414R in subregion 2.2 and substitution T440N in subregion 2.4 change amino acids that are located on the DNA-binding surface of {sigma} (Fig. 3). A mutation of the first residue (K414A) was previously shown to strongly impair open complex formation (21). Two mutations of the second residue, T440I and T440S, affected recognition of the first nucleotide of the –10 element and led to a mild temperature-dependent defect in promoter melting, respectively (6, 7). Thus, these substitutions likely affect the temperature of promoter opening by changing {sigma}-DNA interactions.

In conclusion, we showed that nonconserved amino acids in different subregions of {sigma} region 2 collectively control the temperature of promoter opening through different mechanisms, including changes in protein conformation and flexibility and in contact of {sigma} with core RNAP and DNA. Remarkably, in contrast to previously studied mutations of the conserved amino acids that dramatically impaired transcription initiation, the substitutions studied in our work led to moderate "defects" in open complex formation that resulted specifically in an increase in the temperature of promoter opening. The results of this study show how the evolutionarily conserved transcription mechanism can be fine-tuned by nonconserved amino acid substitutions in functionally important RNAP regions. Comparison of homologous RNAPs and analysis of mosaic enzymes are powerful approaches to identify such changes that underlie lineage-specific differences in transcription.


   ACKNOWLEDGMENTS
 
This work was supported in part by Russian Foundation for Basic Research grant 07-04-00247 and by grant MK-1017.2007.4 from the President of Russian Federation.


   FOOTNOTES
 
* Corresponding author. Mailing address: Laboratory of Molecular Genetics of Microorganisms, Institute of Molecular Genetics, Kurchatov Sq. 2, Moscow 123182, Russia. Phone and fax: 7 (499) 1960015. E-mail: akulb{at}img.ras.ru Back

{triangledown} Published ahead of print on 15 February 2008. Back

{dagger} Supplemental material for this article may be found at http://jb.asm.org/. Back


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Journal of Bacteriology, April 2008, p. 3088-3092, Vol. 190, No. 8
0021-9193/08/$08.00+0     doi:10.1128/JB.00008-08
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