Francisella RNA polymerase contains a heterodimer of non-identical α subunits
© Mukhamedyarov et al; licensee BioMed Central Ltd. 2011
Received: 19 August 2011
Accepted: 22 November 2011
Published: 22 November 2011
All sequenced genomes of representatives of the Francisella genus contain two rpoA genes, which encode non-identical RNA polymerase (RNAP) subunits, α1 and α2. In all other bacteria studied to date, a dimer of identical α subunits initiates the assembly of the catalytically proficient RNAP core (subunit composition α2ββ'). Based on an observation that both α1 and α2 are incorporated into Francisella RNAP, Charity et al. (2007) previously suggested that up to four different species of RNAP core enzyme might form in the same Francisella cell.
By in vitro assembly from fully denatured state, we determined that both Francisella α subunits are required for efficient dimerization; no homodimer formation was detected. Bacterial two-hybrid system analysis likewise indicated strong interactions between the α1 and α2 N-terminal domains (NTDs, responsible for dimerization). NTDs of α2 did not interact detectably, while weak interaction between α1 NTDs was observed. This weak homotypic interaction may explain low-level transcription activity observed in in vitro RNAP reconstitution reactions containing Francisella large subunits (β', β) and α1. No activity was observed with RNAP reconstitution reactions containing α2, while robust transcription activity was detected in reactions containing α1 and α2. Phylogenetic analysis based on RpoA resulted in a tree compatible with standard bacterial taxonomy with both Francisella RpoA branches positioned within γ-proteobacteria. The observed phylogeny and analysis of constrained trees are compatible with Francisella lineage-specific rpoA duplication followed by acceleration of evolutionary rate and subfunctionalization.
The results strongly suggest that most Francisella RNAP contains α heterodimer with a minor subfraction possibly containing α1 homodimer. Comparative sequence analysis suggests that this heterodimer is oriented, in a sense that only one monomer, α1, interacts with the β subunit during the α2β RNAP subassembly formation. Most likely the two rpoA copies in Francisella have emerged through a lineage-specific duplication followed by subfunctionalization of interacting paralogs.
Bioinformatics analysis reveals that two paralogous rpoA genes, each encoding non-identical proteins homologous to bacterial RNA polymerase (RNAP) α subunits, are present in the genome of Francisella tularensis. The bacterial RNAP core enzyme has subunit composition α2ββ'. Variations including fusion of the largest subunits, β and β', in Helicobacter and Wolinella genera [2, 3], and split the largest subunit in some cyanobacteria  have been reported, but overall, the subunit composition of RNAP core is conserved. The α subunit homodimer initiates bacterial RNAP assembly. The α subunit monomers dimerize through their N-terminal domain (NTD) [5, 6]. The C-terminal domain (CTD) is connected to NTD through a flexible tether . The αCTD is not required for assembly but is involved in transcriptional regulation [8–10]. The αNTD homodimer provides a platform for interaction with the two large RNAP subunits [11, 12]. Determinants in α important for interactions with β and β' subunits have been localized by mutagenesis and hydroxyl-radical footprinting studies [5–8, 13–15]. Substitutions at positions 45 and 48 of Escherichia coli α subunit completely (R45A) or partially (L48A) prevented formation of the α2β RNAP subassembly . Two point substitutions at positions 86 and 173, and two-amino-acid insertions at positions 180 and 200 of E. coli α caused defects in β' binding without affecting the α2β assembly formation [16, 17]. RNAP containing oriented E. coli α heterodimers have been prepared both in vitro, by reconstitution from recombinant subunits, and in vivo, by co-expression of genes for recombinant subunits, by using one α subunit lacking the R45A substitution and one α subunit having the R45A substitution [18, 19]. Functional analysis of RNAP containing oriented α heterodimers confirmed that asymmetrical arrangement of α leads to non-identical functions of each monomer in transcription regulation [18, 19].
RNAP core enzymes from archaea and eukaryotes contain homologs of each of the bacterial RNAP core subunits. However, rather than having two identical α subunit homologs, they contain two different α-like polypeptides (RPB3 and RPB11 in the case of eukaryotic RNAP II) that form a heterodimer, which serves as a platform for RNAP assembly .
The presence of two different genes (rpoΑ1 and rpoΑ2) in the genome of Francisella suggests that up to four RNAP core enzymes differing in subunit composition could be present in the cells: two enzymes containing α homodimers, (α1)2ββ' and (α2)2ββ', and two enzymes containing α heterodimers, (α1α2)ββ' and (α2α1)ββ' . The heterodimers could differ from one another with respect to which α interacts with the β subunit of RNAP and which α interacts with β' [18, 19]. Promoter recognition properties of RNAP holoenzymes formed from these different core enzyme molecules may differ, since CTD of α1 and α2 may be capable of different protein-DNA and protein-protein interactions during transcription initiation [18, 19]. Further, if holoenzymes containing RNAP core enzymes of different composition indeed respond differently to transcription factors and elements, then F. tularensis may regulate the spectrum of expressed genes by altering the relative ratio of core enzymes with different α subunit composition, which would be a novel paradigm of transcription regulation in bacteria.
Evidence of that both α1 and α2 subunits are incorporated into F. tularensis RNAP has been reported earlier by Charity et al.. These authors demonstrated that RNAP affinity purified from F. tularensis strain expressing the β' subunit with fused TAP-tag contained both α1 and α2. These experiments clearly show that both rpoA genes are active and their products are components of RNAP but do not inform about the actual subunit composition of F. tularensis RNAP. Based on predicted dimerization determinants in other bacteria , Charity et al. hypothesized that α1 and α2 might exclusively form either homodimers or heterodimers . In the present study, we describe the results of in vitro analysis of assembly of RNAP from F. tularensis subspecies novicida. Our results indicate that RNAP core containing an α heterodimer is the main, perhaps the only, species of RNAP in this organism. We further present results of phylogenetic analysis that provide a plausible scenario for the appearance of two paralogous rpoA genes in the Francisella lineage.
F. tularensis α heterodimer but not homodimers efficiently assembles in vitro
Therefore, because each of the four proteins used in the pull-down assay has a characteristic electrophoretic mobility, it is possible to detect the efficiency of both hetero- and homodimer formation. Various pairwise combinations of α subunits were mixed at denaturing conditions (6 M guanidinium chloride), the denaturing reagent was removed by dialysis at conditions favouring bacterial RNAP assembly from isolated subunits , and reconstitution reactions (labelled "L" on Figure 1) were loaded on Ni2+-affinity columns. Flow-through (F) was collected and retained protein was eluted (E) with different concentrations of imidazole in the buffer. Aliquots of each fraction were next analyzed by SDS-PAGE. As can be seen from Figure 1 (top panel), no co-immobilization of untagged α subunit in reactions that contained tagged and untagged versions of subunit of the same kind was detected. In contrast, heterodimers were readily detected when either α1His6 or α2His6 were used as "baits" for co-immobilization of, respectively, α2 or α1 (Figure 1, bottom panel). We conclude that F. tularensis RNAP α subunits do not appreciably form homodimers, at least at the conditions of in vitro RNAP assembly.
α1NTD and α2NTD efficiently interact in bacterial 2-hybrid system
Bacterial two-hybrid analysis of interactions between domains of α1 and α2 subunits of F. tularensis RNAP
171 ± 28
44 ± 1
1412 ± 323
53 ± 9
110 ± 13
95 ± 8
133 ± 5
99 ± 16
662 ± 26
47 ± 9
74 ± 4
49 ± 3
102 ± 3
101 ± 1
112 ± 6
86 ± 9
Formation of the α2β subassembly in vitro
In vitro transcription by recombinant F. tularensis RNAP
Evolution of RpoA in Francisella
To gain insight into the evolution of two paralogs of RpoA in Francisella, we retrieved all RpoA sequences in all of the 1055 completely sequenced bacterial genomes available in the RefSeq database. We found that in addition to Francisella, two rpoA genes (rpoA1 and rpoA2) are present in several other genomes, namely in three Chloroflexus species (C. aggregans DSM 9485; C. aurantiacus J-10-fl; C. sp. Y-400-fl), in Streptomyces avermitilis MA-4680, in Psychromonas ingrahamii 37, and in Leptospira borgpetersenii serovar Hardjo bovis L550 (see also Additional file 1). In the latter two cases the two rpoA copies are identical and are apparently the result of very recent genome segment duplications that also include a number of other genes.
Representatives of the bacterial genus Francisella are unusual with respect to RNAP composition, in that they contain two different α subunits encoded by two paralogous genes rpoΑ1 and rpoΑ2. The presence of two different α subunits in affinity-purified RNAP preparations  suggested that as many as four different species of RNAP core enzyme could be present in the single cell. Here, we studied Franicella RNAP by means of in vitro assembly. Our results show that Franicella α heterodimer (α1α2) efficiently assembles in vitro from fully denatured state and homodimers are not detected. Bacterial two-hybrid analysis indicates that in addition to efficient α heterodimer assembly, some dimerization of α1 may also occur. Thus, the efficiency of α dimerization is clearly a major factor that should affect subunit composition of Francisella RNAP. As was determined from crystal structure analysis, the main structural elements of the α dimer interface of E. coli are two α-helices, H1 and H3, orthogonally oriented to each other . These helices from one monomer participate in a coiled-coil-like interaction with their counterparts in the other monomer. Within these helices, Kannan et al.  identified a cluster of amino acids stabilizing interactions at the E. coli α dimer interface, with residues 35F, 38T, and 39L emanating from one α monomer and residues 46I, 50S, and 227Q from another. Of particular interest are three of them, namely, 35F, 38T, and 46I, point mutations at which partially (α-T38A)  or completely (α-F35A, α-I46S)  prevented the dimer formation. As can be seen from Figure 2B, amino acids at these positions are conserved (identical) between E. coli and Thermus, while amino acids in many of the corresponding positions in both α1 and α2 subunits from Francisella differ. Since these positions are critical for the dimer formation, it is reasonable to assume that some amino acids at these positions of Francisella α subunits, for example, α1-36M, α1-39I, α2-33V, and α2-47T, may be unfavourable to the assembly of homodimers. However, in the absence of crystal structure or systematic mutagenesis data, it is currently not possible to identify structural reasons for hetero- and homodimerization of Francisella α subunits.
During RNAP assembly in organisms where α subunit forms a homodimer, the β subunit is free to interact with either α monomer to form the α2β subassembly. The situation must be different in the case of F. tularensis, where α heterodimers form preferentially. In E. coli, an evolutionary conserved α subunit residue Arg45 is critical for β subunit interaction with the α dimer [16, 18, 19]. In F. tularensis, the corresponding position in α1 also contains an arginine (Arg46), while in α2 this position is occupied by glutamine (Gln42; Figure 2B). Thus, it appears that β subunit in F. tularensis will be specifically interacting with the α heterodimer through α1. That α1 contains determinants for interactions with β also follows from results of the α2β subassembly reconstitution and in vitro transcription data, since Francisella RNAP containing α1 homodimer is functional and formation of an (α1)2β intermediate can be detected in vitro, albeit with low efficiency. The latter result suggests that β may stimulate α1 dimerization. An alternative possibility would be β interacting with one α1 monomer, followed by association with another α1 and β', or the α1β' complex. Be that as it may, our data suggest that bacteria of Francisella genus produce a major form of RNAP containing an oriented α1α2 heterodimer, and a minor form containing α1 homodimer.
As shown earlier by hydroxyl-radical-mediated proteolysis , the segments of E. coli α most strongly protected by β correspond to amino acids 30-55 and 65-75, and the segments of α most strongly protected by β' correspond to amino acids 175-185 and 195-210. Single alanine substitutions of E. coli α Lys86 and Val173 and two-amino-acid insertions at positions 180 and 200 of E. coli α cause defects in β' binding without affecting the α2β assembly formation [16, 17]. To evaluate the ability of Francisella α1 and α2 subunits to interact with the β' subunit, we compared sequences of E. coli α subunit involved in interaction with β' [14, 21] to those in F. tularensis α1 and α2 subunits (Additional file 1). The results reveal that a lysine at a position corresponding to E. coli α position 86 is present in both α polypeptides from Francisella, while amino acids corresponding to E. coli α Val173 are, respectively, a valine and a leucine in α1 and α2. Similarly, the site of one two-amino-acid insertion that destroys β' interaction with α2β in E. coli, Val180, has as its counterpart a valine in α2 and an isoleucine in α1 of Francisella. These conservative changes are unlikely to affect the efficiency of β' binding by the α polypeptides. Interestingly, the site of the residue at the site of the second insertion affecting β' interaction with α2β in E. coli, Lys200, is conserved in Francisella α2 and α subunits from other bacteria (see Additional file 1), but is substituted with threonine in α1. The results thus implies that Francisella β' interacts with α2. Further experiments will be needed to prove this conjecture.
Our phylogenetic analysis indicates that none of the Francisella rpoA genes was transferred from outside of the y-proteobacterial lineage. In fact, both genes most likely emerged through duplication of an ancestral single gene followed by acceleration of evolutionary rate of both paralogs. Acceleration of rpoA evolutionary rate after the duplication apparently was accompanied by subfunctionalization of Francisella α subunits, ultimately leading to accumulation of substitutions in residues responsible for homodimerization and involved in the (β and β' subunit interaction.
Similar events, albeit on much longer time intervals, must have led to formation of two very different α-like subunits in eukaryotes and archaea. The large α-like subunit (RPB3 in eukaryal RNAP II, AC40 in RNAP I and RNAP III, Rpo3 (also known as RpoD) in archaea) heterodimerizes with its much smaller counterpart (RPB11 in eukaryal RNAP II, AC19 in RNAP I and RNAP III, Rpo11 (also known as RpoL) in archaea) [33–36]. Crystallographic and functional analyses indicates that large α homolog makes interaction with the second-largest (β-like) subunit through a surface that contains residue homologous to E. coli α Arg45[35, 37], and is thus formally similar to Francisella α1. The smaller α homolog of eukaryal and archaeal RNAP thus corresponds to Francisella α2. One should not take this analogy to far though, since in eukaryotes and archaea, the α heterodimer is not sufficient for recruitment of large RNAP subunits in the complex. Eukaryal RPB10 and RPB12 and their archaeal homologs Rpo10 (also known as RpoN) and Rpo12 (also known as RpoP) form a stable complex with all four polypeptides playing an essential role in assembly and stability of the RNAP complex [36, 38, 39].
Evolution of the rpoA duplication presented here is one of the best demonstrations in support of the Lynch's subfunctionalization scenario where both copies are subject to relaxed selection and acceleration of the evolutionary rates but rarely develop a new or specialized function . The fact that bacterial RNAP α subunit functions as a dimer should make it particularly prone to duplication/subfunctionalization. Indeed, while the impetus for our study came from an apparently unique situation with two different α subunits in Francisella, bioinformatics analysis revealed additional instances of rpoA duplications, some fairly recent, like in S. avermitilis, others more ancient, like in Chloroflexus species. Despite the fact that these RpoA paralogs are being ancestral for the Chloroflexus species, no drastic substitutions in regions responsible for dimerization and/or β/β' interactions have accumulated, suggesting that in contrast to the situation observed in Fransicella, the two α subunits of Chloroflexus may still be functionally equivalent. It is likely that many more instances of rpoA duplications and subfunctionalization will be found in the future.
The data presented here support the following conclusions: (1) only Francisella α-heterodimer (α1α2) can be efficiently assembled in vitro; (2) strong direct interactions between α1NTD and α2NTD only have been detected in the bacterial two-hybrid system; (3) β interacts more efficiently when both of α1 and α2 presented in the reconstitution mix; (4) interaction between α1 and β subunits was observed to be stronger than interaction between α2 and β; (5) based on phylogenetic analysis, two rpoA copies in Francisella most likely must have emerged through a duplication followed by acceleration of the evolutionary rates of both paralogs.
E. coli NovaBlue Singles competent cells (Novagen) were used for initial cloning and plasmid propagation. E. coli BL21 (DE3) cells were used for protein overproduction. Reporter E. coli strain FW102 F'OL2-62  was used for bacterial two-hybrid experiments.
Cloning and expression
Francisella tularensis novicida genomic DNA has been provided by Dr. Michael Ibba (Ohio State University). Primers for PCR amplification of rpo genes were designed using Francisella tularensis subsp. novicida (FTN) strain U112 genome sequence data [GenBank: NC_008601]. The primers allowed cloning of amplified FTN rpo genes in pET series E. coli expression plasmids between the NcoI and EcoRI (or XhoI to express C-terminally hexahistidine-tagged α subunits) restriction sites. The plasmids pET28-FtnA1His, pET28-FtnA2His, pET28-FtnA1, pET28-FtnA2, and pET28-FtnB, pET30-FtnC overexpressing, respectively, C-terminally hexahistidine-tagged αlHis6 and α2His6, and untagged α1, α2, and β, and N-terminally hexahistidine-tagged β' subunits, were constructed using routine cloning methods and verified by sequencing of entire rpo portions for each plasmid. BL21 (DE3) cells harbouring the pET28a-rpo-gene plasmids were grown in 500 ml of LB medium, supplemented with 25 μg/ml kanamycin, at 37°C until an OD600 of around 1 was reached. Then the culture was induced to express an RNAP subunit by the addition of 1 mM isopropyl β-D-1-thiogalactopyranoside and allowed to grow for 2-4 hours. Cells were harvested by centrifugation and stored at -80°C before use.
Frozen cells were thawed and lysed by sonication in a buffer containing 40 mM Tris-HCl, pH 8.0, 0.1 M NaCl, 10 mM EDTA, 10 mM 2-ME. The lysate was clarified by centrifugation for 30 min at 15,000 × g, and supernatant was supplemented with ammonium sulphate to precipitate soluble proteins (α2, α1His6, α2His6). Pellets containing insoluble proteins (α1, β, α1His6, α2His6, His6-β') were resuspended in 40 mM Tris-HCl, pH 8.0, 0.3 M KCl, 10 mM EDTA, 0.2% Na-deoxycholate to prepare inclusion bodies as described before [22, 42]. Ammonium sulphate precipitate was collected by centrifugation, and the pellet, containing untagged protein (α2), was dissolved in 40 mM Tris-HCl, pH 8.0, 0.1 M NaCl, 1 mM EDTA, 2 mM 2-ME, 0.1 mM PMSF and loaded onto a 1 ml HiTrap Heparin HP column (GE Healthcare) equilibrated in the same buffer. The bound protein was eluted by a linear gradient of NaCl (from 0.1 to 1.0 M) in the same buffer. The pellet containing α1His6 or α2His6 was dissolved in 20 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 2 mM 2-ME, 0.1 mM PMSF, 5 mM imidazole and loaded onto the 1 ml HiTrap Chelating HP column (GE Healthcare) charged with Ni2+. The column was washed and bound protein was step-eluted with 20, 50, or 100 mM imidazole. Fractions containing pure protein were pooled and dialyzed against two changes of 500 volumes of 40 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.5 mM EDTA, 0.5 mM 2-ME, 10% glycerol, concentrated by ultrafiltration using Microsep (PALL, Life Science) centrifugal device, and stored at -80°C.
In vitro protein interaction experiments
Purified proteins were mixed together in pairwise combinations of untagged and his-tagged proteins. Before mixing, proteins in inclusion bodies were solubilised in denaturing buffer containing 6M guanidine-HCl, 20 mM Tris-HCl, pH 8, 10 mM MgCl2, 10 μM ZnCl2, 1 mM EDTA, 10mM DTT, 10% glycerol. Coupled proteins were mixed in 0.5-1 ml of denaturing buffer at equimolar ratio and adjusted to 0.2-0.5 mg/ml total protein concentration. Refolding of denatured molecules was achieved by removing guanidine-HCl from reaction mix through one-change dialysis against 500 ml of reconstitution buffer (20 mM Tris-HCl, pH 8, 0.2 M NaCl, 10 mM MgCl2, 10 μM ZnCl2, 0.25 mM EDTA, 0.5 mM DTT, 20% glycerol). Precipitate formed during dialysis was removed by centrifugation. Then supernatant was diluted 4-fold with start buffer (25 mM HEPES, pH 8.0, 0.5 M NaCl, 5% glycerol, 1 mM imidazole) and loaded on 0.5 ml His-Select Nickel Affinity Gel (Sigma) column equilibrated in the same buffer. The column was washed with start buffer, and proteins were eluted with three steps of start buffer containing 20, 100, 200 mM imidazole. Fractions were analyzed by SDS-PAGE and visualized by Coomassie-staining.
Reconstitution of α2β, and α2ββ' was performed as described above. The α2 β subassemblies were stepwise fractionated on Ni2+-affinity column with 10, 50, and 100 mM imidazole; α2ββ' RNAP core assembly reactions were fractionated by gel-filtration on a Superose 6 column (GE Healthcare) in the buffer containing 40 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, and 1 mM 2-ME. Superose 6 fractions were checked for transcription activity on a nucleic acid scaffold. For this purpose, nucleic acid scaffold containing radioactively labelled 8-nt RNA primer (Figure 3A) was added into 10 μl of the target Superose 6 fraction to obtain artificial transcription elongation complexes and transcription was initiated by the addition of NTP and Mg2+. Reaction products were resolved by denaturing 20% PAGE and revealed by autoradiography.
Bacterial two-hybrid assays
Gene fragments encoding Francisella α1NTD (residues 1-244), α2NTD (residues 1-242), α1CTD (residues 227-323), α2CTD (residues 226-318), were cloned in the plasmids pBRαLN and pACλcI to gain fusions with E.coli RNAP αNTD and bacteriophage λ cI proteins respectively [24, 43, 44]. FW102 F'OL2-62 reporter strain cells were co-transformed with every possible pairwise plasmid combination. Individual transformants were selected and grown in 2 ml of LB medium supplemented with 50 μg/ml carbenicillin, 25 μg/ml kanamycin, 25 μg/ml chloramphenicol, and 0.1 mM IPTG. β-galactosidase assays were performed as described earlier .
RpoA comparative analysis
For comparative analysis of two RpoA paralogs in Francisella we retrieved the RefSeq database (NCBI) containing 1055 completely sequenced bacterial genomes on March 2010. A set of 368 RpoA sequences from 355 representative genomes was aligned with MUSCLE program , and the maximum likelihood tree for 246 informative aligned positions was built using FastTree program . RAxML program  was used for reconstruction of the phylogenetic tree for γ-proteobacteria with β-proteobacteria as an outgroup. The same program was used for comparison of the maximum likelihood values for the best and constrained trees. The evolutionary model for tree reconstitution (WAG  with gamma-distributed evolutionary rates) was selected with ProtTest program .
The authors thank Y. Wolf and S. Nechaev for critical reading of the manuscript and useful discussions. The project was supported by NIAID Award AI090558 to KK. KSM is supported by the intramural funds of the US Department of Health and Human Services (National Library of Medicine). This work was partially supported by an NIH GM59295 grant and Russian Academy of Sciences Presidium program grant in Molecular and Cellular Biology to KS.
- Charity JC, Costante-Hamm MM, Balon EL, Boyd DH, Rubin EJ, Dove SL: Twin RNA polymerase-associated proteins control virulence gene expression in Francisella tularensis. PLoS Pathog. 2007, 3 (6): e84- 10.1371/journal.ppat.0030084PubMed CentralView ArticlePubMedGoogle Scholar
- Zakharova N, Hoffman PS, Berg DE, Severinov K: The largest subunits of RNA polymerase from gastric helicobacters are tethered. J Biol Chem. 1998, 273 (31): 19371-19374. 10.1074/jbc.273.31.19371View ArticlePubMedGoogle Scholar
- Zakharova N, Paster BJ, Wesley I, Dewhirst FE, Berg DE, Severinov KV: Fused and overlapping rpoB and rpoC genes in Helicobacters, Campylobacters, and related bacteria. J Bacteriol. 1999, 181 (12): 3857-3859.PubMed CentralPubMedGoogle Scholar
- Bergsland KJ, Haselkorn R: Evolutionary relationships among eubacteria, cyanobacteria, and chloroplasts: evidence from the rpoC1 gene of Anabaena sp. strain PCC 7120. J Bacteriol. 1991, 173 (11): 3446-3455.PubMed CentralPubMedGoogle Scholar
- Igarashi K, Fujita N, Ishihama A: Identification of a subunit assembly domain in the alpha subunit of Escherichia coli RNA polymerase. J Mol Biol. 1991, 218 (1): 1-6. 10.1016/0022-2836(91)90865-4View ArticlePubMedGoogle Scholar
- Hayward RS, Igarashi K, Ishihama A: Functional specialization within the alpha-subunit of Escherichia coli RNA polymerase. J Mol Biol. 1991, 221 (1): 23-29. 10.1016/0022-2836(91)80197-3View ArticlePubMedGoogle Scholar
- Blatter EE, Ross W, Tang H, Gourse RL, Ebright RH: Domain organization of RNA polymerase alpha subunit: C-terminal 85 amino acids constitute a domain capable of dimerization and DNA binding. Cell. 1994, 78 (5): 889-896. 10.1016/S0092-8674(94)90682-3View ArticlePubMedGoogle Scholar
- Igarashi K, Ishihama A: Bipartite functional map of the E. coli RNA polymerase alpha subunit: involvement of the C-terminal region in transcription activation by cAMP-CRP. Cell. 1991, 65 (6): 1015-1022. 10.1016/0092-8674(91)90553-BView ArticlePubMedGoogle Scholar
- Ross W, Gosink KK, Salomon J, Igarashi K, Zou C, Ishihama A, Severinov K, Gourse RL: A third recognition element in bacterial promoters: DNA binding by the alpha subunit of RNA polymerase. Science. 1993, 262 (5138): 1407-1413. 10.1126/science.8248780View ArticlePubMedGoogle Scholar
- Ishihama A: Protein-protein communication within the transcription apparatus. J Bacteriol. 1993, 175 (9): 2483-2489.PubMed CentralPubMedGoogle Scholar
- Zillig W, Palm P, Heil A: Function and Reassembly of Subunits of DNA-dependent RNA Polymerase. RNA Polymerase. Edited by: R Losick & M Chamberlin. 1976, 6: 101-125. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press,Google Scholar
- Ishihama A: Subunit of assembly of Escherichia coli RNA polymerase. Adv Biophys. 1981, 14: 1-35.PubMedGoogle Scholar
- Kimura M, Fujita N, Ishihama A: Functional map of the alpha subunit of Escherichia coli RNA polymerase. Deletion analysis of the amino-terminal assembly domain. J Mol Biol. 1994, 242 (2): 107-115. 10.1006/jmbi.1994.1562View ArticlePubMedGoogle Scholar
- Heyduk T, Heyduk E, Severinov K, Tang H, Ebright RH: Determinants of RNA polymerase alpha subunit for interaction with beta, beta', and sigma subunits: hydroxyl-radical protein footprinting. Proc Natl Acad Sci USA. 1996, 93 (19): 10162-10166. 10.1073/pnas.93.19.10162PubMed CentralView ArticlePubMedGoogle Scholar
- Igarashi K, Fujita N, Ishihama A: Sequence analysis of two temperature-sensitive mutations in the alpha subunit gene (rpoA) of Escherichia coli RNA polymerase. Nucleic Acids Res. 1990, 18 (20): 5945-5948. 10.1093/nar/18.20.5945PubMed CentralView ArticlePubMedGoogle Scholar
- Kimura M, Ishihama A: Functional map of the alpha subunit of Escherichia coli RNA polymerase: amino acid substitution within the amino-terminal assembly domain. J Mol Biol. 1995, 254 (3): 342-349. 10.1006/jmbi.1995.0621View ArticlePubMedGoogle Scholar
- Kimura M, Ishihama A: Functional map of the alpha subunit of Escherichia coli RNA polymerase: insertion analysis of the amino-terminal assembly domain. J Mol Biol. 1995, 248 (4): 756-767. 10.1006/jmbi.1995.0258View ArticlePubMedGoogle Scholar
- Murakami K, Kimura M, Owens JT, Meares CF, Ishihama A: The two alpha subunits of Escherichia coli RNA polymerase are asymmetrically arranged and contact different halves of the DNA upstream element. Proc Natl Acad Sci USA. 1997, 94 (5): 1709-1714. 10.1073/pnas.94.5.1709PubMed CentralView ArticlePubMedGoogle Scholar
- Estrem ST, Ross W, Gaal T, Chen ZW, Niu W, Ebright RH, Gourse RL: Bacterial promoter architecture: subsite structure of UP elements and interactions with the carboxy-terminal domain of the RNA polymerase alpha subunit. Genes Dev. 1999, 13 (16): 2134-2147. 10.1101/gad.13.16.2134PubMed CentralView ArticlePubMedGoogle Scholar
- Cramer P: Multisubunit RNA polymerases. Curr Opin Struct Biol. 2002, 12 (1): 89-97. 10.1016/S0959-440X(02)00294-4View ArticlePubMedGoogle Scholar
- Zhang G, Darst SA: Structure of the Escherichia coli RNA polymerase alpha subunit amino-terminal domain. Science. 1998, 281 (5374): 262-266.View ArticlePubMedGoogle Scholar
- Borukhov S, Goldfarb A: Recombinant Escherichia coli RNA polymerase: purification of individually overexpressed subunits and in vitro assembly. Protein Expr Purif. 1993, 4 (6): 503-511. 10.1006/prep.1993.1066View ArticlePubMedGoogle Scholar
- Ebright RH, Busby S: The Escherichia coli RNA polymerase alpha subunit: structure and function. Curr Opin Genet Dev. 1995, 5 (2): 197-203. 10.1016/0959-437X(95)80008-5View ArticlePubMedGoogle Scholar
- Dove SL, Joung JK, Hochschild A: Activation of prokaryotic transcription through arbitrary protein-protein contacts. Nature. 1997, 386 (6625): 627-630. 10.1038/386627a0View ArticlePubMedGoogle Scholar
- Zalenskaya K, Lee J, Gujuluva CN, Shin YK, Slutsky M, Goldfarb A: Recombinant RNA polymerase: inducible overexpression, purification and assembly of Escherichia coli rpo gene products. Gene. 1990, 89 (1): 7-12. 10.1016/0378-1119(90)90199-2View ArticlePubMedGoogle Scholar
- Tang H, Severinov K, Goldfarb A, Ebright RH: Rapid RNA polymerase genetics: one-day, no-column preparation of reconstituted recombinant Escherichia coli RNA polymerase. Proc Natl Acad Sci USA. 1995, 92 (11): 4902-4906. 10.1073/pnas.92.11.4902PubMed CentralView ArticlePubMedGoogle Scholar
- Korzheva N, Mustaev A, Kozlov M, Malhotra A, Nikiforov V, Goldfarb A, Darst SA: A structural model of transcription elongation. Science. 2000, 289 (5479): 619-625. 10.1126/science.289.5479.619View ArticlePubMedGoogle Scholar
- Battistuzzi FU, Feijao A, Hedges SB: A genomic timescale of prokaryote evolution: insights into the origin of methanogenesis, phototrophy, and the colonization of land. BMC Evol Biol. 2004, 4: 44- 10.1186/1471-2148-4-44PubMed CentralView ArticlePubMedGoogle Scholar
- Ciccarelli FD, Doerks T, von Mering C, Creevey CJ, Snel B, Bork P: Toward automatic reconstruction of a highly resolved tree of life. Science. 2006, 311 (5765): 1283-1287. 10.1126/science.1123061View ArticlePubMedGoogle Scholar
- Park HK, Yoon JW, Shin JW, Kim JY, Kim W: rpoA is a useful gene for identification and classification of Streptococcus pneumoniae from the closely related viridans group streptococci. FEMS Microbiol Lett. 2010, 305 (1): 58-64. 10.1111/j.1574-6968.2010.01913.xView ArticlePubMedGoogle Scholar
- Stamatakis A: RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics. 2006, 22 (21): 2688-2690. 10.1093/bioinformatics/btl446View ArticlePubMedGoogle Scholar
- Kannan N, Chander P, Ghosh P, Vishveshwara S, Chatterji D: Stabilizing interactions in the dimer interface of alpha-subunit in Escherichia coli RNA polymerase: a graph spectral and point mutation study. Protein Sci. 2001, 10 (1): 46-54. 10.1110/ps.26201PubMed CentralView ArticlePubMedGoogle Scholar
- Lalo D, Carles C, Sentenac A, Thuriaux P: Interactions between three common subunits of yeast RNA polymerases I and III. Proc Natl Acad Sci USA. 1993, 90 (12): 5524-5528. 10.1073/pnas.90.12.5524PubMed CentralView ArticlePubMedGoogle Scholar
- Larkin RM, Guilfoyle TJ: Reconstitution of yeast and Arabidopsis RNA polymerase alpha-like subunit heterodimers. J Biol Chem. 1997, 272 (19): 12824-12830. 10.1074/jbc.272.19.12824View ArticlePubMedGoogle Scholar
- Naryshkina T, Rogulja D, Golub L, Severinov K: Inter- and intrasubunit interactions during the formation of RNA polymerase assembly intermediate. J Biol Chem. 2000, 275 (40): 31183-31190.View ArticlePubMedGoogle Scholar
- Werner F, Weinzierl RO: A recombinant RNA polymerase II-like enzyme capable of promoter-specific transcription. Mol Cell. 2002, 10 (3): 635-646. 10.1016/S1097-2765(02)00629-9View ArticlePubMedGoogle Scholar
- Cramer P, Bushnell DA, Fu J, Gnatt AL, Maier-Davis B, Thompson NE, Burgess RR, Edwards AM, David PR, Kornberg RD: Architecture of RNA polymerase II and implications for the transcription mechanism. Science. 2000, 288 (5466): 640-649. 10.1126/science.288.5466.640View ArticlePubMedGoogle Scholar
- Werner F, Grohmann D: Evolution of multisubunit RNA polymerases in the three domains of life. Nat Rev Microbiol. 9 (2): 85-98.View ArticleGoogle Scholar
- Werner F, Eloranta JJ, Weinzierl RO: Archaeal RNA polymerase subunits F and P are bona fide homologs of eukaryotic RPB4 and RPB12. Nucleic Acids Res. 2000, 28 (21): 4299-4305. 10.1093/nar/28.21.4299PubMed CentralView ArticlePubMedGoogle Scholar
- Lynch M, Conery JS: The evolutionary demography of duplicate genes. J Struct Funct Genomics. 2003, 3 (1-4): 35-44.View ArticlePubMedGoogle Scholar
- Deaconescu AM, Chambers AL, Smith AJ, Nickels BE, Hochschild A, Savery NJ, Darst SA: Structural basis for bacterial transcription-coupled DNA repair. Cell. 2006, 124 (3): 507-520. 10.1016/j.cell.2005.11.045View ArticlePubMedGoogle Scholar
- Tang H, Kim Y, Severinov K, Goldfarb A, Ebright RH: Escherichia coli RNA polymerase holoenzyme: rapid reconstitution from recombinant alpha, beta, beta', and sigma subunits. Methods Enzymol. 1996, 273: 130-134.View ArticlePubMedGoogle Scholar
- Dove SL, Hochschild A: A bacterial two-hybrid system based on transcription activation. Methods Mol Biol. 2004, 261: 231-246.PubMedGoogle Scholar
- Hu JC, Kornacker MG, Hochschild A: Escherichia coli one- and two-hybrid systems for the analysis and identification of protein-protein interactions. Methods. 2000, 20 (1): 80-94. 10.1006/meth.1999.0908View ArticlePubMedGoogle Scholar
- Edgar RC: MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics. 2004, 5: 113 10.1186/1471-2105-5-113PubMed CentralView ArticlePubMedGoogle Scholar
- Price MN, Dehal PS, Arkin AP: FastTree 2--approximately maximum-likelihood trees for large alignments. PLoS One. 2010, 5 (3): e9490- 10.1371/journal.pone.0009490PubMed CentralView ArticlePubMedGoogle Scholar
- Whelan S, Goldman N: A general empirical model of protein evolution derived from multiple protein families using a maximum-likelihood approach. Mol Biol Evol. 2001, 18 (5): 691-699.View ArticlePubMedGoogle Scholar
- Darriba D, Taboada GL, Doallo R, Posada D: ProtTest 3: fast selection of best-fit models of protein evolution. Bioinformatics. 2011, 27 (8): 1164-1165. 10.1093/bioinformatics/btr088View ArticlePubMedGoogle Scholar
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