Inactivation of the Bacterial RNA Polymerase Due to Acquisition of Secondary Structure by the ω Subunit*

Background: Although universally conserved, ω is the only nonessential subunit of RNA polymerase, and its function is mostly undetermined. Results: Dominant lethal mutants of ω were isolated and analyzed genetically and biochemically. Conclusion: Mutations in ω that alter its structure interfere with RNA polymerase catalytic properties. Significance: Results suggest a critical role for ω during transcription, bypassing the effects of compensatory proteins. The widely conserved ω subunit encoded by rpoZ is the smallest subunit of Escherichia coli RNA polymerase (RNAP) but is dispensable for bacterial growth. Function of ω is known to be substituted by GroEL in ω-null strain, which thus does not exhibit a discernable phenotype. In this work, we report isolation of ω variants whose expression in vivo leads to a dominant lethal phenotype. Studies show that in contrast to ω, which is largely unstructured, ω mutants display substantial acquisition of secondary structure. By detailed study with one of the mutants, ω6 bearing N60D substitution, the mechanism of lethality has been deciphered. Biochemical analysis reveals that ω6 binds to β′ subunit in vitro with greater affinity than that of ω. The reconstituted RNAP holoenzyme in the presence of ω6 in vitro is defective in transcription initiation. Formation of a faulty RNAP in the presence of mutant ω results in death of the cell. Furthermore, lethality of ω6 is relieved in cells expressing the rpoC2112 allele encoding β′2112, a variant β′ bearing Y457S substitution, immediately adjacent to the β′ catalytic center. Our results suggest that the enhanced ω6-β′ interaction may perturb the plasticity of the RNAP active center, implicating a role for ω and its flexible state.

The widely conserved subunit encoded by rpoZ is the smallest subunit of Escherichia coli RNA polymerase (RNAP) but is dispensable for bacterial growth. Function of is known to be substituted by GroEL in -null strain, which thus does not exhibit a discernable phenotype. In this work, we report isolation of variants whose expression in vivo leads to a dominant lethal phenotype. Studies show that in contrast to , which is largely unstructured, mutants display substantial acquisition of secondary structure. By detailed study with one of the mutants, 6 bearing N60D substitution, the mechanism of lethality has been deciphered. Biochemical analysis reveals that 6 binds to ␤ subunit in vitro with greater affinity than that of . The reconstituted RNAP holoenzyme in the presence of 6 in vitro is defective in transcription initiation. Formation of a faulty RNAP in the presence of mutant results in death of the cell. Furthermore, lethality of 6 is relieved in cells expressing the rpoC2112 allele encoding ␤ 2112 , a variant ␤ bearing Y457S substitution, immediately adjacent to the ␤ catalytic center. Our results suggest that the enhanced 6 -␤ interaction may perturb the plasticity of the RNAP active center, implicating a role for and its flexible state.
The bacterial RNAP 2 holoenzyme is a multisubunit complex that performs the essential function of gene transcription (1,2). In Escherichia coli, the core of RNAP contains two ␣ subunits (␣I and ␣II) and three distinct subunits designated as ␤, ␤Ј, and (3)(4)(5)(6). The core RNAP associates with one of the various alternative subunits to form the holo-RNAP to initiate transcription from specific promoter sequences in the bacterial genome (7,8).
Although extensive information is available on the contribution of various ␤, ␤Ј, and ␣ subunits in the primary functioning of bacterial RNA polymerase (9,10), the biological role of the subunit is less clear (11). The subunit encoded by the rpoZ gene is the smallest constituent of RNAP with a molecular mass of 10 kDa and is found in near stoichiometric amounts in preparations of both core and holo-RNAP from E. coli (3,12). Although identified in the initial years of RNAP research, was not considered to be a bona fide subunit mainly because of two major observations. In vivo deletion of rpoZ is tolerated in bacterial cells (13) unlike deletion of the other subunits of RNAP. In addition, reconstitution of E. coli RNAP was achieved with purified ␣, ␤, and ␤Ј subunits (14), thus pointing at the redundancy of .
However, two experiments in the last decade allowed us to contemplate for the role of . First, tethering experiment demonstrated that when is covalently linked to a DNA-binding protein it is able to activate transcription (15). On the other hand, the crystal structures of Thermus aquaticus RNAP (5) and that of E. coli RNAP published recently (16) confirm the presence of as a subunit of RNAP beyond ambiguity. Such contention was further supported by the analysis of the sequences of several bacterial genomes where the presence of rpoZ was noticed in addition to its similarity to a subunit of eukaryotic RNAP, Rpb6 (17), and archaeal RNAP, RpoK (18).
Previously we had observed that RNAP isolated from a -less strain of E. coli co-purifies with global chaperone GroEL, and attempts to remove GroEL ultimately destroy the enzyme (19). This indicates, albeit tenuously, that acts like a chaperone for RNAP. Interestingly, when the crystal structure was determined (5), it was mentioned that the arrangement of vis-à-vis ␤Ј subunit was such that its role as a chaperone is conceivable. The subunit cross-links exclusively with ␤Ј subunit in RNAP (20), and its association with the ␤Ј subunit promotes the binding of -␤Ј with ␣ 2 ␤ subassembly (21). An analysis of the T. aquaticus RNAP core combined with functional studies has further defined the features of the /␤Ј interface in the RNAP core (17), leading to the proposal that may promote assembly and/or stability of ␤Ј in RNAP by functioning as a clamp that latches the N-terminal half of ␤Ј to its C terminus to conforma-tionally constrain ␤Ј. The surface of subunit interacts extensively with the double ␤-barrel (DPBB) domain of ␤Ј subunit (22), which makes up the catalytic center of RNAP, suggesting an important role of subunit assembly for RNAP catalysis. Intriguingly, the E. coli RNAP crystal reveals an alternative conformation of the C-terminal tail that is very different from that of T. aquaticus RNAP (17), indicating a possible differential regulatory aspect of that has yet to be deciphered.
Other than its role in mediating effective assembly of RNAP, a physiological link between and the stringent response has been reported. This suggestion is based on the observation that in vitro a -less RNAP is rendered non-responsive to ppGpp, the effector of the stringent response, and that the presence of DksA, a coeffector (23), or externally added (24) rescues this defect. In addition, in strains lacking , expression of the relA promoter is impaired (25). Furthermore, recent crystal structures of the RNAP-ppGpp complex and a biochemical study showed that ppGpp binds at the interface between ␤Ј and subunits, and the N terminus of is involved in transcription regulation mediated by ppGpp (26 -28).
In this study, we used an alternative approach to delineate the function exclusive to . The genetic screen used acts to counter the influences of factors like GroEL and DksA known to elicit compensatory effects and complicate the elucidation of functionality. We have thus isolated a set of rpoZ variants whose conditional expression in vivo is associated with lethality. Apart from the lethality, all the mutant proteins exhibit a unique feature of structural reorganization into a more compact conformation. Studies with one variant, 6 , show its enhanced association with ␤Ј without impeding the assembly of RNAP but resulting in inactivation of RNAP. As we probed into the various steps of transcription, it is evident that the enzyme with mutant is defective in a step of transcription initiation subsequent to the initial binding to the promoter. On a preformed elongation complex in the presence of RNA primer, the enzyme can catalyze transcription. Results from extragenic suppressor and in vitro reconstitution studies suggest that structural flexibility of is crucial for maintaining an active RNAP catalytic center. Changes that reorient to a more structured entity possibly affect RNAP plasticity by holding the DPBB catalytic domain of ␤Ј in an unfavorable conformation.

MATERIALS AND METHODS
Bacterial Strains-All strain used in the study have been listed in Table 1. The bacterial strains DH5␣, MG1655, and PD1 constructed by phage-based transduction (29) are derivatives of E. coli K12 and were used in genetic studies. All strains were propagated on Luria Bertani (LB) broth and agar. The rpoZ::kan allele was sourced from the strain JW3624 (30). The BL21(DE3) strain (31) was used for protein overproduction and purification. The antibiotics ampicillin and spectinomycin were used for selection of plasmid-borne antibiotic resistance phenotypes, namely Amp R and Spec R , respectively, at appropriate concentrations, and D-glucose and L-arabinose were used for repressing and inducing, respectively, the P ara promoter (32) at the indicated concentrations.

Isolation of Dominant Lethal rpoZ Variants-
The open reading frame (ORF) encoding , rpoZ ϩ (wild type), was PCR-amplified from the chromosome of the strain MG1655 with a primer pair (5Ј-TGTGGAGCTTTTTACATATGGCACGAG-TAACTTCAGCAATAG-3Ј and 5Ј-ACAAGGGCGACCCGC-TAAGCTTTTAACGACGACCTTCAGCAATAG-3Ј) and was cloned into the NdeI and HindIII sites of pHYD3011 (modified pBAD18 (32)) to generate plasmid pRPZ rendering the expres- sion of rpoZ ϩ under the control of the P ara promoter. With the genomic DNA of strain MG1655 as a template and the above mentioned primers, we generated pools of PCR-amplified rpoZ amplicons using error-prone PCR utilizing alterations in PCR conditions (33)(34)(35)(36). The combined pool of rpoZ PCR amplicons was cloned into pHYD3011 (selectable marker Amp R ), and the ligation mixture was transformed into DH5␣. Transformants were isolated on LB agar plates supplemented with ampicillin and 0.2% D-glucose, and 2000 transformants were individually tested for their inability to grow on LB plates with ampicillin and 0.2% L-arabinose. Those that did not grow in L-arabinose medium but grew well on D-glucose medium were considered as bearing plasmids that expressed potential dominant lethal versions of rpoZ. Using plasmid DNA isolated from the counterpart colony on D-glucose LB plates, we tested by retransformation that the phenotype of dominant lethality was plasmidborne. The rpoZ ORF from the chosen transformants was cloned into the vector pHYD3011 and sequenced after establishing the dominant lethal phenotype. The N60D substitution was recreated on the rpoZ ϩ ORF by site-directed mutagenesis using the megaprimer method (37). The sequences of mutagenic primer and flanking primer are 5Ј-GAAGGTCTGATC-GACAACCACATC-3Ј and 5Ј-AAGCTTTTAACGACG-3Ј, respectively. The mutagenized PCR amplicon was cloned in pHYD3011 identically to wild-type and mutagenized amplicon pools.
Total Cellular RNA Isolation-Primary cultures of DH5␣ cells bearing pRPZ and pRPZM6 were grown in the presence of 0.02% glucose for 5 h at 37°C in LB. 1% of the primary culture was washed and subcultured in 0.02% L-arabinose in LB and grown for 3 h. A 600 was normalized for the two cultures. Total cellular RNA was isolated using an RNeasy Mini kit (Qiagen). The RNA was checked by 1.2% formaldehyde agarose gel electrophoresis. For the uninduced sample, primary culture was subcultured in 0.02% D-glucose-containing LB.
-␤Ј and 6 -␤Ј Association Studies-Strain BL21(DE3) was used for protein preparation. Plasmids pRPZ and pRPZM6 were transformed in BL21(DE3) cells. Cultures of the transformed derivatives were grown to an A 600 of 0.6, induced with 0.002% L-arabinose for 6 -8 h at 37°C, and purified using the protocol described previously (12). Plasmid pRW308 (38) was used for overproduction and purification of ␤Ј using a previously published protocol (39). 6 -␤Ј complex formation was assessed by both immunoprecipitation and gel-based experiments. Purified proteins were mixed in a 1:1 molar ratio in denaturation buffer (6 M urea, 50 mM Tris HCl, pH 7.9, 200 mM KCl, 10 mM MgCl 2 , 10 mM ZnCl 2 , 10% glycerol, 1 mM EDTA, 10 mM DTT added fresh) and incubated for 1 h at 4°C. The total protein concentration was kept at 0.5 mg ml Ϫ1 . The mixture was then dialyzed against reconstitution buffer (50 mM Tris-HCI, pH 7.9, 300 mM KCl, 10 mM MgCl 2 , 10 M ZnCl 2 , 0.1 mM EDTA, 1 mM DTT added fresh, 20% glycerol) at 4°C to allow the formation of the complex. The dialyzed sample was spun at 10,000 ϫ g at 4°C for 15 min to settle debris, and the supernatant containing the complex was collected.
To detect protein-protein interaction, the supernatant containing 6 -␤Ј complex was electrophoresed on a 6% Tris borate-polyacrylamide gel. Western blot with anti-antibody identified the 6 -␤Ј complex species in a parallel gel run. A gel slice cut from the former gel identified to be 6 -␤Ј complex based upon migration of the immunodetected signal was minced, boiled with SDS loading buffer, and loaded onto an 8 -20% gradient SDS-polyacrylamide gel. The protein species corresponding to 6 and ␤Ј were identified by silver staining as well as Western blot. The interaction was also confirmed by immunoprecipitation (data not shown).
Surface Plasmon Resonance (SPR) Studies-SPR studies to obtain quantitative estimates of -␤Ј and 6 -␤Ј associations were carried out in a Biacore 3000 instrument at 25°C. Purified ␤Ј was immobilized on a CM5 chip according to the manufacturer's recommendations. Solutions of both and 6 were flowed over the chip separately in running buffer (10 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA). Binding experiments used samples of at least four different concentrations of and 6 . After every injection of or 6 , the sensor chip surface was regenerated by rapid injection of 2-10 l of 0.01-0.04% SDS. The data were analyzed globally by fitting both the association and dissociation phases simultaneously (BIAevaluation software, version 4.1) to a 1:1 (Langmuir) model. Uncertainties were calculated using three sets of curves for each of the two samples with each set comprising four curves of various concentrations of and 6 .
Isolation of the rpoC2112 Suppressor Allele-Plasmid pHYD535 is a derivative of the plasmid pCL1920 bearing the pSC101 replicon (40) and contains a 10.2-kb HindIII DNA fragment bearing the rplL ϩ and the rpoB ϩ C ϩ genes sourced from plasmid pDJJ12 (41). pHYD535 was subjected to in vitro hydroxylamine mutagenesis (42), and the pool of mutagenized plasmid DNA was used to transform DH5␣ cells bearing pRPZM6. L-Arabinose-resistant (Ara R ) transformants able to survive the L-arabinose-induced lethality of rpoZM6 were selected on LB plates containing ampicillin, spectinomycin (for selection of the Spec R marker of pHYD535), and 0.02% L-arabinose.
One Ara R transformant was isolated, and its Ara R phenotype co-segregated with the Spec R marker on pHYD535 in DH5␣ cells bearing the plasmid pRPZM6. The plasmid so recovered was designated pRPC1 and is a derivative of pHYD535. DNA sequencing of the 10.2-kb insert in pRPC1 revealed a base alteration that would create Y457S substitution in rpoC ϩ (rpoC2112).
The Y457S amino acid substitution in ␤Ј was created by sitedirected mutagenesis of rpoC on plasmid pHYD535 using a primer pair (5Ј-CTGGTTTGTGCGGCATCTAACGCCGACT-TCGATGGT-3Ј and 5Ј-CCGGCAGCGGATTGTGCTAGCTC-CGACGGGAGCAAATCCGTGAAAGATTTATTA-3Ј) with high fidelity Phusion (Thermo Scientific) DNA polymerase, and the amino acid substitution was verified by DNA sequencing. From pRPC1, rpoC2112 was amplified with a primer pair (5Ј-CCGGCAGCGGATTGTGCTAGCTCCGACGGGAGC-AAATCCGTGAAAGATTTATTA-3Ј and 5Ј-GTTTTTACG-TTATTTGCGGATTAACTCGAGCTCGTTATCAGAACC-GCCCAGACC-3Ј) and cloned into the BamHI-XhoI site of pET21b to generate plasmid pRPC3, which allows for overproduction of ␤Ј 2112 -C-His 6 polypeptide. The same primer pair was used to clone ␤Ј ORF identically in pET21b, generating pRPC2 to allow for overproduction of ␤Ј-C-His 6 . All PCRs described in this section were done using high fidelity Phusion (Thermo Scientific) DNA polymerase.
Promoter DNA Binding-An electrophoretic mobility shift assay for DNA binding was performed using an 85-bp 5Ј-GAATTCAATTTAAAAGAGTATTGACTTAAAGTCTAA-CCTATAGGATACTTACAGCCAGAGAGAGGGAGAAG-GGAATCGGGGATCC-3Ј double-stranded DNA fragment bearing T7A1 promoter. Promoter DNA was end-labeled using ␥-32 P-labeled ATP by polynucleotide kinase. Polymerase binding to detect the stable complex with promoter was carried out in a reaction mixture containing 45 mM Tris borate, 5 mM magnesium acetate, 0.1 mM DTT, 50 mM KCl, 50 g ml Ϫ1 BSA, 5% glycerol, 2 pmol of labeled DNA, and polymerase in a 10-l reaction for 15 min at 30°C. Heparin was added to the mixture at a final concentration of 50 g ml Ϫ1 to remove any nonspecific DNA-polymerase complex. The reaction products were separated in a 4% non-denaturing polyacrylamide gel.
In Vitro Gel-based Single Round Transcription Assay-The abortive transcription assay to detect first nucleotide bond formation was done as described (47). The single round in vitro gel-based transcription assay was performed as reported previously (48). Plasmid pAR1432 was used for the preparation of T7A1 promoter DNA template (49).
Reconstitution of Elongation Complex and Determining Its Transcriptional Activity-For in vitro reconstitution of elongation complex, the 30-nucleotide-long template strand and 9-mer RNA scaffold were designed as described previously (50). The reconstitution of elongation complex and transcription assay were carried out as described before (50).

Isolation of Dominant Lethal Variants and Rationale of the
Genetic Screen-This work originated from our observation that -less RNAP has some inherent weakness that is not apparent in vivo as it secures protection from GroEL. Consequently, any phenotypic peculiarity that appears to be associated with mutational variation in gets masked by the global chaperone. We reasoned that if played a role in one or more steps of the RNAP transcription cycle, including those mediating promoter binding, opening, escape, RNA chain initiation, and elongation, then a mutation in that interfered with any of such essential steps might produce a lethal phenotype that would occur even in the presence of the wild-type rpoZ (encoding ) allele.
To isolate such dominant lethal variants of , we generated libraries of rpoZ products obtained after in vitro PCR mutagenesis. The PCR products were cloned under the transcriptional control of a P ara promoter-bearing plasmid, rendering expression of cloned rpoZ variants conditional. This screen allows selection of variants where the wild-type protein gets titrated by the faulty counterpart in vivo and GroEL is prevented from eliciting its rescue function on RNAP. The expression of dominant lethal rpoZ variants would be repressed in the presence of D-glucose (leading to their isolation) and induced in the presence of L-arabinose (32). A population of transformants of the strain DH5␣ bearing a library of PCR-mutagenized rpoZ genes was isolated in the presence of D-glucose in LB medium. Each transformant was screened for its inability to grow in the presence of L-arabinose. Such mutants were found at a frequency of 0.75%. We sequenced the rpoZ genes of four plasmids bearing dominant lethal rpoZ mutants whose conditional expression by L-arabinose at concentrations ranging from 0.002 to 0.2% produced a marked lethality. Most of the mutations were found to be clustered in the C-terminal tail region of the protein. One plasmid bore a base alteration in rpoZ leading to an N60D amino acid substitution in (pRPZM6; encoding 6 ), whereas produced from the other two plasmids contained an A85T (pRPZM15; encoding 15 ) substitution and a double D36Y and N60D substitution (pRPZM2; encoding 2 ) ( Table 2). We also isolated a dominant lethal variant bearing a silent third base alteration (GCC3 GCT) marking generation of a rare codon for the 82nd amino acid (pRPZM9; encoding 9 ).
Properties of the 6 Variant-Because the N60D substitution was isolated twice and independently, we studied the properties of bearing this alteration, designated as 6 , in greater detail. Although the expression of rpoZ ϩ in the presence of L-arabinose produced no discernable alterations in growth phenotypes in the genetic backgrounds of DH5␣, MG1655, and PD1 (MG1655 rpoZ::kan), expression of rpoZM6 (encoding 6 ) yielded a dominant lethal phenotype in the above mentioned genetic backgrounds (Fig. 1, A, B, and C). The N60D amino acid substitution was recreated by site-specific mutagenesis of rpoZ ϩ to yield a similar dominant lethal phenotype. In addition, upon expression from the P ara promoter, the levels of and 6 were comparable on an SDS-polyacrylamide gel (data not shown). The N60D alteration occurs in the CR3 conserved region of (17) (Fig. 1D).

TABLE 2 List of variants indicating the resident nucleotide change and corresponding amino acid change of each mutated rpoZ
The percentage of ␣helicity of each protein calculated with K2D2 software from their CD profile has been tabulated.

Nucleotide change
Amino acid change ␣-Helicity % Wild-type 15 6 A3G N60D  82  2  G3T  D36Y  80  A3G  N60D  15  C3T  A85T  60  9 G3A Silent mutation 61 The growth profile of DH5␣/pRMZ6 in the presence of L-arabinose shows a marked reduction in cell viability ( Fig. 2A). In an attempt to understand the extreme weakness of the mutants, the total RNA was isolated from L-arabinose (0.02%)-induced DH5␣/pRZM6 cells. The total cellular RNA was also extracted from DH5␣ bearing pRPZ grown in L-arabinose as control. The A 600 values of the induced cultures were normalized to have equal amounts of cells prior to RNA isolation. The comparative RNA profile indicates a decrease of the mRNA pool upon induction (Fig. 2B). This result implicated a possible defect in the transcription machinery in the mutant strain that eventually leads to cell death.
Acquisition of Extensive Secondary Structure by 6 -The mutant protein 6 (Fig. 3A) bearing N60D alteration results in an increment in molecular mass of by 0.98 -1 Da that we have tested and verified in an independent study involving mass spectrometry (MS) (51). MS analysis also ensured that the isolated 6 was 100% mutant variety with no detectable contamination of the wild-type (51). 6 was found to be stable compared with , which tends to precipitate upon storage at 4°C. Far-UV CD spectra of and 6 revealed that 6 had acquired extensive secondary structure in comparison with . The estimated percentage of ␣-helicity using K2D2 (52) software was 82% for 6 and 15% for (Fig. 3B and Table 2).
As mentioned earlier, associates with the ␤Ј subunit of RNAP ( Fig. 3D and supplemental Movie 1). Thus, the structural alteration of 6 prompted us to check whether the 6 -␤Ј interaction remained unaltered. To this effect, purified ␤Ј and 6 protein were allowed to reconstitute, any complex formed was tested in a gel-based assay wherein a preformed 6 -␤Ј complex  was subjected to electrophoresis in a non-denaturing 6% polyacrylamide gel (Fig. 4A), and a similar gel run in parallel was probed with antiantibody (Fig. 4B). The protein band corresponding to the position of the Western signal was excised from the former gel, and its constituents were separated and detected on an 8 -20% gradient SDS-polyacrylamide gel and identified appropriately by antiand anti-␤Ј antibodies (Fig. 4, C and D). The 6 -␤Ј interaction was further validated by an immunoprecipitation experiment showing that mutant associates as successfully with ␤Ј as its wild-type counterpart (data not shown).
The -␤Ј and 6 -␤Ј associations were further compared quantitatively by SPR studies wherein a range of concentrations of both proteins were flowed over immobilized ␤Ј. SPR analyses indicated that 6 possessed ϳ10-fold higher affinity for ␤Ј as compared with with a significant increment in its rate of association (Table 3). It is worth mentioning that the SPR study shows a very small association rate constant (k a ), which is indicative of a very large conformational change in the ␤Ј. This is expected because it is known that confers stability to ␤Ј upon interaction.
Reconstitution of RNAP with 6 -We reconstituted the holo-RNAP bearing and 6 (RNAP and 6 RNAP, respectively) in vitro (see "Materials and Methods") to monitor RNAP assembly and observed that both core and holoenzyme can be reconstituted in the presence of and 6 (Fig. 5, A and B).
To ascertain whether the reconstituted 6 RNAP binds to promoter DNA, an electrophoretic mobility shift assay was performed. Radiolabeled T7A1 promoter DNA fragment (85 bp) was incubated with RNAP enzyme preparations, and complex formation was checked. 6 RNAP was found to form heparinresistant stable complexes with labeled promoter fragment comparable with those formed by the reconstituted RNAP (Fig. 5C). An RNAP Preparation Bearing 6 Is Defective in Transcription Initiation-The transcriptional activity of the reconstituted RNAP preparations was tested in an in vitro heparinresistant single round transcription assay. 6 RNAP was found to be inactive in mediating transcription at the T7A1 promoter (Fig. 5D). On the other hand, RNAP exhibited activity comparable with that displayed by an RNAP preparation isolated from the bacterial cell (Fig. 5D). 6 RNAP was found to be defective in the formation of the first phosphodiester bond in the transcription assay when only the first and the second (radiolabeled) nucleotides were provided (Fig. 5E), leading to the notion that 6 RNAP is defective for initiation of transcription. On a preformed elongation complex where the first few nucleotides were provided in the form of RNA primer, 6 RNAP was able to resume transcription (Fig. 5F).
Lethality of 6 Is Suppressed in Cells Expressing the rpoC 2112 Suppressor Mutation Encoding ␤Ј 2112 -To further the understanding of cellular lethality of 6 , we sought to obtain extragenic suppressors in gene(s) encoding RNAP subunits that may potentially suppress the cellular lethality imposed by the expression of rpoZM6. Our search for an extragenic suppressor was somewhat directed given the altered association of 6 with ␤Ј. For this purpose, we subjected the rpoB ϩ C ϩ genes cloned in pHYD535, a derivative of the low copy (pSC101 replicon) plasmid pCL1920, to hydroxylamine mutagenesis. In pHYD535, the expression of the rplL ϩ and rpoB ϩ C ϩ genes that are located within a 10.2-kb HindIII fragment sourced in turn from the plasmid pDJJ12 (41) is expected to occur from the plasmidborne P lac promoter. Nevertheless, the presence of plasmid pHYD535 rescued the thermosensitive growth phenotype of a strain bearing an rpoC(ts) mutation (data not shown). In this suppressor search, the suppressor mutation is expected to exert its phenotypic effect in a dominant manner to overcome the lethality of 6 and in turn yield an L-arabinose-resistant (Ara R ) growth phenotype with the chromosomal rpoB ϩ C ϩ genes behaving as neutral genetic entities. The presence of plasmid pHYD535 on its own does not yield an Ara R phenotype (Fig.  6A). Following hydroxylamine mutagenesis of plasmid pHYD535 and the transformation of this pool of plasmids into DH5␣ bearing plasmid pRPZM6, the source of P ara -expressing 6 , we selected for transformants that may survive the lethality imposed by the L-arabinose-induced production of 6 under appropriate L-arabinose-inducing conditions and antibiotic selection on LB agar plates. We isolated a transformant exhib-  iting a weak Ara R phenotype indicative of partial suppression. The L-arabinose-induced lethality conferred by rpoZM6 is seen with L-arabinose concentrations ranging from 0.002 to 0.2%, and at lower concentrations of L-arabinose (0.002-0.1%), the Ara R phenotype of the said transformant was marked (Fig. 6A). Because the Ara R phenotype in the co-transformant segregated with the antibiotic marker of plasmid pHYD535, we sequenced the entire rpoB ϩ C ϩ insert DNA present on the pHYD535 derivative (pRPC1). We found that pRPC1 bore a base alteration in the rpoC gene leading to Y457S amino acid substitution in rpoC (Fig. 6, A and B) occurring adjacent to the 458 NAD-FDGD 464 active site motif of ␤Ј that chelates the catalytic Mg 2ϩ metals (53,54). A possible reason for the partial suppression (Ara R ) phenotype conferred by pRPC1 is that the levels of RpoB and RpoC in plasmid pHYD535 (and pRPC1) may be limiting because the rpoBC genes are under heterologous expression control differing from that found in their chromosomal context. Recreation of the cognate base alteration in the rpoC gene present in pRPC1 led to a similar regeneration of the Ara R phenotype conferred by pRPC1 (data not shown). The suppressor rpoC allele was further cloned into pET21b (Novagen) to introduce a His 6 tag for ease of purification devoid of contamination from the chromosomal wild-type ␤Ј. The suppressor rpoC allele and its corresponding protein are referred to as rpoC 2112 and ␤Ј 2112 , respectively.
Reconstituted 6 RNAP Bearing the ␤Ј 2112 Subunit Is Transcriptionally Active in Vitro-Using purified ␤Ј and ␤Ј 2112 subunits in addition to ␣, wild-type , and 70 subunits, we gauged the transcription capacities of reconstituted holo-RNAP preparations with in vitro single round gel-based transcription assays. 6 RNAP bearing the ␤Ј 2112 subunit in contrast to 6 RNAP bearing the ␤Ј subunit regained rifampicin-sensitive transcription activity at the T7A1 promoter template (Fig. 7A). In comparative transcriptional profiling (Fig. 7, B and C), RNAP bearing the ␤Ј 2112 or ␤Ј subunit displayed a transcriptional efficiency similar to that of the 6 -␤Ј 2112 RNAP preparation.
Gain of Structure Is Common Feature of All Lethal Variants-We obtained far-UV CD spectra of other dominant lethal variants isolated in this study and found that in general all variants displayed a gain of helical content ( Table 2). To our knowledge, such a result is unprecedented. This result indicates that not only are the WT residues at these positions somehow responsible for the unstructured state of the wild-type protein but also that each of these mutant residues favors folding (or assembly into multimers?) to impart a large structural change to the protein.

Correlation between Helical Content and Toxicity of Mutant
Variants-Because all mutant variants that have been selected through the genetic screen show increment of helical content to different degrees, we tried to determine whether there is any correlation between the extent of toxicity and the percentage of helicity gained by the mutants. As the phenotype being scored in the genetic screen is lethality or absence of growth, establishing any direct quantitative correlation can be challenging. To this end, we performed an indirect genetic assessment. Plasmids bearing two of the mutants that show different helical content, 6 (82%) and 15 (60%), were introduced in wild-type DH5␣. The cells were grown in the presence of L-arabinose (0.02%) until the A 600 reached 0.2, allowing expression of the toxic protein. The cultures were then washed twice with LB, equal amount of cells were used to inoculate fresh LB with 0.2% glucose, and recovery of the cells as a function of time was monitored. We found that cells expressing more helical ( 6 ) recovered at a slower rate (Fig. 8). From the figure, it can be seen that the change in rate of growth between the two mutants is small; however, they are consistent over several experiments. This indicates that the more helical is comparatively more toxic than the less helical variant.

DISCUSSION
Decades of extensive research have given us a clearer picture of how RNAP machinery functions, but the role of its smallest constituent subunit has remained controversial (11). Declared redundant from the very beginning, has resurfaced now and again with intriguing observations, proclaiming the need for renewed investigation.
The Unusual Phenotype of a Dominant Lethal rpoZ Mutation-The phenotype resulting from the conditional expression of rpoZM6 (encoding 6 ) is one that is counterintuitive because a deficiency of does not overtly affect bacterial growth (Fig. 1,  A, B, and C). The physiological perturbation caused by 6 , therefore, must occur at a step(s) that is essential for bacterial viability, in this case gene transcription. In general, dominant lethal mutations can occur both in proteins that are essential and in those that participate in an essential cellular process in an apparently redundant manner. However, they all possess one of two properties: (a) either the mutant proteins in some manner interfere with the functioning of their wild-type counterparts or (b) they exert their effects directly on the essential process. For example, aspartate to alanine replacements in the conserved NADFDGD active site residues of the ␤Ј subunit produce a dominant lethal phenotype (53,54) thought to occur because of increased persistence of open complex formation by the mutant RNAP that impedes the action of wild-type RNAP. On the other hand, the E96D substitution in RecA (RecA*), a nonessential protein, produces a dominant lethal phenotype (55) that is postulated to occur because the impaired RecA* DNA dissociation impedes essential DNA transactions. Such mutations have also been referred to as toxic mutations (55), and the 6 mutant isolated in this study falls in the RecA* type category.
The toxic mutant isolation strategy used here where plasmid-based overexpression of the protein is used to detect phenotypic alteration can be argued as an artificial situation inside the cell. Because chromosomal deletion of rpoZ does not elicit any phenotype, this system appears to be promising and one of the few options to study the exclusive role of . The lack of phenotype in the rpoZ-null strain also prohibited us to move the mutations in the chromosome. The reduction in the total RNA profile, which is marked by the reduction of mRNA synthesis (Fig. 2B), specifically highlights that in vivo global transcription of the cell has been affected due to the mutation. 6 Binds to ␤Ј with Greater Affinity than -The route to obtaining an answer to the general question "why is 6 toxic to the cell?" probably begins with our observation that the dissociation constant between 6 and ␤Ј is significantly lower than that between and ␤Ј, suggesting that the structural transition in 6 is causal to this effect ( Table 3). The similarity between this observation and the two examples of dominant lethality cited (53,55) is striking. In all three cases, the genesis of the lethal event occurs via shifting the equilibrium of a naturally occurring transient interaction toward a more stable association by the mutant protein. Nevertheless, these studies suggest that, in addition to promoting the assembly of ␤Ј (19), may play a second role(s) once it is lodged in the confines of the RNAP core. 6 -mediated Catalytic Inactivation of RNAP-As one of the major functions of is to assemble ␣ 2 ␤ subcomplex with properly folded ␤Ј subunit (19), we were undoubtedly surprised to note that 6 did not impair the assembly of RNAP holoenzyme (Fig. 5A). We detected the association of 70 in 6 RNAP, suggesting that the lethal effect of 6 is not due to a loss of 70 -6 RNAP core interactions (Fig. 5B). As we dissected each step in the transcription initiation by 6 RNAP, it became clear that the enzyme retains its ability to form promoter complex (Fig.  5C) but is unable to initiate transcription (Fig. 5E) and thus rendered defective (Figs. 5D and 7C). When the initiation step is bypassed, the protein is able regain its transcriptional activity. This explanation for the lethality is further strengthened by the isolation of the rpoC2112 (encoding ␤Ј 2112 ) extragenic suppressor whose presence provides a substantial bypass of both 6 lethality in vivo (Fig. 6A) and 6 -mediated blockade of tran-scription in vitro (Fig. 7A). Approximately 60% of the transcriptional activity was recovered compared with reconstituted wild-type RNAP (Fig. 7C). Interestingly, ␤Ј 2112 bears the Y457S substitution located immediately adjacent to the conserved 458 NADFDGD 464 active site residues located on ␤Ј that coordinates the catalytic Mg 2ϩ metals ( Fig. 6B and supplemental Movie 1).
A Structural Transition in 6 Is Critical to Its Lethality-Perhaps the most striking correlate of the pathology of 6 is the remarkable structural transition (Fig. 3B) it bears caused by N60D substitution. This result suggests that biological RNAP activity becomes in some manner sensitive to a large scale alteration in the intrinsic protein disorder present in . Analyses of domain structure of by limited proteolysis (21) have shown that possesses a structured N-terminal region corresponding to the first 40 -50 amino acids of comprising the CR1 and CR2 domains (17) (Fig. 1D). Using current nomenclature that includes proteins of partial structural disorder (56 -58), can be bracketed in the category of an intrinsically disordered protein.
The CD profiles of all dominant lethal variants show acquisition of substantial secondary structure ( Table 2). The nature of the variants is quite remarkable considering that all mutants have a single mutation (except 2 ) and show extensive structural alteration. Intriguingly, we also isolated a silent mutation in rpoZ (GCC-GCT at the 82nd codon of rpoZ) whose expression caused a dominant lethal phenotype, and remarkably, its cognate protein also showed increment in structural content compared with (Table 2). This seemingly perplexing observation can be rationalized as an example of a synonymous single nucleotide polymorphism, which marks substitution of an amino acid codon with its rare counterpart. As has been reported (59), this change may alter the conformation due to slowdown of the protein synthesis rate, hence influencing its catalytic property. This observation strongly suggests a general mechanism whereby ordering of the unstructured tail results in lethality. It is plausible that the ordered proteins may function through inactivation of RNAP beyond promoter binding as established for 6 . The observation also highlights the importance of the unstructured nature of . The intrinsic disorder in proteins and their functional significance have been a subject of considerable interest (60 -62). Chaperone proteins in particular are known to possess interspersed unstructured elements (63). It has been suggested that flexibility provided by these elements is key to their role (63). The chaperone activity provided by to the largest subunit serves as an example of this phenomenon.
Implications for the Physiological Role of -Detailed structure analysis of the /␤Ј interface in E. coli RNAP (17) has revealed that a globular N-terminal domain of (residues 2-59) makes contacts with ␤Ј subunit in four regions, namely regions D and G (residues 905-913), residues 614 -615, and a region proximal to the terminus (residues 1360 -1361), whereas the C-terminal extended ␣-helices of (from residue 61 to the C terminus) does not interact with ␤Ј subunit (Fig. 3, C and D, and supplemental Movie 1). The Asn 60 residue of is positioned at a junction between these two domains and within a cavity of the N-terminal domain, suggesting that the N60D mutation changes amino acid interactions of the N-terminal domain for acquisition of secondary structure of .
Region D of ␤Ј forms the DPBB domain, which contains the NADFDGD active site residues to enable chelation of Mg 2ϩ in the RNAP active center (22,54). The DPBB domain also makes extensive contacts with , but its binding surface is located at the side opposite the RNAP active site. It is worth mentioning that the interface between and ␤Ј regulates RNAP transcription both positively and negatively depending on promoters and factors (64). In addition, the DPBB domain is a hot spot of compensatory mutations for reducing the fitness cost associated with rifampicin-resistant mutations of Mycobacterial tuberculosis RNAP (65).
The catalytic site architectural specificities ␤-D, ␤-H, ␤Ј-D, and ␤Ј-G form distinct domains that surround the central motif (Fig. 6B). Mutational studies supported by cross-linking experiments suggest that distortion of the catalytic site results in varying degrees of inactivation of the enzyme (43). It has been well established that ␤Ј-G along with region F constitutes the flexible/mobile ensemble of the RNAP catalytic center mediating the placement of cognate NTPs and unidirectional RNA extension at the RNAP active center (66 -68). The inactivated polymerase that is reconstituted in the presence of 6 retains its ability to bind promoter segment, but subsequent steps prior to elongation are impaired (Fig. 5, C, D, E, and F). Thus, we postulate that the large structural alteration in 6 may affect the flexibility of the ␤Ј mobile elements in the RNAP catalytic center needed for transcription initiation and act as a transcriptional inhibitor. It appears that the C-terminal end of subunit is important for regulation. If subunit is acting as a receiver of signals triggered by binding of a small molecule like ppGpp (26 -28) or structural change as in this case, then the difference between the stringent response elicited by T. aquaticus enzyme vis-à-vis that of E. coli may be explained. We further speculate that might act in a modular fashion and transmit the molecular signal via the DPBB region to the active site. On the other hand, it is possible that 6 may directly interfere with the functioning of the RNAP catalytic center or of another RNAP subunit. In addition, a previous finding that overproduction of mediates suppression (17) of the thermosensitive growth phenotype of a certain (but not all) ts allele of rpoC (rpoC tsX ) that encodes an extremely unstable ␤Ј subunit appears to be consistent with the present observation that -␤Ј maintains active site plasticity. In this regard, the survival of a strain lacking may be explained on the basis that in its absence the associated GroEL (19) prevents a total structural collapse possibly of ␤Ј and that new stability acquired by ␤Ј leads to reduced transcriptional activity because of compromised plasticity of the RNAP catalytic center.