X-ray Crystal Structure of Escherichia coli RNA Polymerase σ70 Holoenzyme

Escherichia coli RNA polymerase (RNAP) is the most studied bacterial RNAP and has been used as the model RNAP for screening and evaluating potential RNAP-targeting antibiotics. However, the x-ray crystal structure of E. coli RNAP has been limited to individual domains. Here, I report the x-ray structure of the E. coli RNAP σ⁷⁰ holoenzyme, which shows σ region 1.1 (σ_1.1) and the α subunit C-terminal domain for the first time in the context of an intact RNAP. σ_1.1 is positioned at the RNAP DNA-binding channel and completely blocks DNA entry to the RNAP active site. The structure reveals that σ_1.1 contains a basic patch on its surface, which may play an important role in DNA interaction to facilitate open promoter complex formation. The α subunit C-terminal domain is positioned next to σ domain 4 with a fully stretched linker between the N- and C-terminal domains. E. coli RNAP crystals can be prepared from a convenient overexpression system, allowing further structural studies of bacterial RNAP mutants, including functionally deficient and antibiotic-resistant RNAPs.     

Stimulation of human DNA polymerase epsilon by MDM2

The human DNA polymerase ε catalytic subunit consists of a 140-kDa N‐terminal domain that contains the catalytic activity and a 120-kDa C-terminal domain that binds to the other subunits and to exogenous peptides, including PCNA and MDM2. We report here that recombinant human MDM2 purified from insect cells or Escherichia coli stimulated the activity of DNA polymerase ε up to 10- and 40-fold, respectively, but not those of DNA polymerase β or Klenow fragment of E.coli DNA polymerase I. Kinetic studies indicated that MDM2 increased the maximum velocity of the reaction, but did not change substrate affinities. The stimulation depended upon the interaction of the N‐terminal 166 amino acid residues of MDM2 with the C-terminal domain of the full-length catalytic subunit, since the deletion of 166 amino acids from N‐terminal of MDM2 or the removal of the C-terminal domain of DNA polymerase ε by trypsin digestion or competition for binding to it by the addition of excess C-terminal fragment eliminated the stimulation. Since DNA polymerase ε appears to be involved in DNA replication, recombination and repair synthesis, we suggest that MDM2 binding to DNA polymerase ε might be part of a reconfiguration process that allows DNA polymerase ε to associate with repair/recombination proteins in response to DNA damage.     

The DNA polymerase III holoenzyme contains γ and is not a trimeric polymerase

There is widespread agreement that the clamp loader of the Escherichia coli replicase has the composition DnaX₃δδ’χψ. Two DnaX proteins exist in E. coli, full length τ and a truncated γ that is created by ribosomal frameshifting. τ binds DNA polymerase III tightly; γ does not. There is a controversy as to whether or not DNA polymerase III holoenzyme (Pol III HE) contains γ. A three-τ form of Pol III HE would contain three Pol IIIs. Proponents of the three-τ hypothesis have claimed that γ found in Pol III HE might be a proteolysis product of τ. To resolve this controversy, we constructed a strain that expressed only τ from a mutated chromosomal dnaX. γ containing a C-terminal biotinylation tag (γ-C_tag) was provided in trans at physiological levels from a plasmid. A 2000-fold purification of Pol III* (all Pol III HE subunits except β) from this strain contained one molecule of γ-C_tag per Pol III* assembly, indicating that the dominant form of Pol III* in cells is Pol III₂τ₂ γδδ’χψ. Revealing a role for γ in cells, mutants that express only τ display sensitivity to ultraviolet light and reduction in DNA Pol IV-dependent mutagenesis associated with double-strand-break repair, and impaired maintenance of an F’ episome.     

Only One ATP-binding DnaX Subunit Is Required for Initiation Complex Formation by the Escherichia coli DNA Polymerase III Holoenzyme

The DnaX complex (DnaX₃δδ′χψ) within the Escherichia coli DNA polymerase III holoenzyme serves to load the dimeric sliding clamp processivity factor, β₂, onto DNA. The complex contains three DnaX subunits, which occur in two forms: τ and the shorter γ, produced by translational frameshifting. Ten forms of E. coli DnaX complex containing all possible combinations of wild-type or a Walker A motif K51E variant τ or γ have been reconstituted and rigorously purified. DnaX complexes containing three DnaX K51E subunits do not bind ATP. Comparison of their ability to support formation of initiation complexes, as measured by processive replication by the DNA polymerase III holoenzyme, indicates a minimal requirement for one ATP-binding DnaX subunit. DnaX complexes containing two mutant DnaX subunits support DNA synthesis at about two-thirds the level of their wild-type counterparts. β₂ binding (determined functionally) is diminished 12–30-fold for DnaX complexes containing two K51E subunits, suggesting that multiple ATPs must be bound to place the DnaX complex into a conformation with maximal affinity for β₂. DNA synthesis activity can be restored by increased concentrations of β₂. In contrast, severe defects in ATP hydrolysis are observed upon introduction of a single K51E DnaX subunit. Thus, ATP binding, hydrolysis, and the ability to form initiation complexes are not tightly coupled. These results suggest that although ATP hydrolysis likely enhances β₂ loading, it is not absolutely required in a mechanistic sense for formation of functional initiation complexes.     

Protection of Salmonella by ampicillin-resistant Escherichia coli in the presence of otherwise lethal drug concentrations

Microbial systems have become the preferred testing grounds for experimental work on the evolution of traits that benefit other group members. This work, based on conceptual and theoretical models of frequency-dependent selection within populations, has proven fruitful in terms of understanding the dynamics of group beneficial or ‘public goods’ traits within species. Here, we expand the scope of microbial work on the evolution of group-beneficial traits to the case of multi-species communities, particularly those that affect human health. We examined whether β-lactamase-producing Escherichia coli could protect ampicillin-sensitive cohorts of other species, particularly species that could cause human disease. Both β-lactamase-secreting E. coli and, surprisingly, those engineered to retain it, allowed for survival of a large number of ampicillin-sensitive cohorts of Salmonella enterica serovar Typhimurium, including both laboratory and clinical isolates. The Salmonella survivors, however, remained sensitive to ampicillin when re-plated onto solid medium and there was no evidence of gene transfer. Salmonella survival did not even require direct physical contact with the resistant E. coli. The observed phenomenon appears to involve increased release of β-lactamase from the E. coli when present with S. enterica. Significantly, these findings imply that resistant E. coli, that are not themselves pathogenic, may be exploited, even when they are normally selfish with respect to other E. coli. Thus, Salmonella can gain protection against antibiotics from E. coli without gene transfer, a phenomenon not previously known. As a consequence, antibiotic-resistant E. coli can play a decisive role in the survival of a species that causes disease and may thereby interfere with successful treatment.     

Effects of DNA3′pp5′G capping on 3′ end repair reactions and of an embedded pyrophosphate-linked guanylate on ribonucleotide surveillance

When DNA breakage results in a 3′-PO₄ terminus, the end is considered ‘dirty’ because it cannot prime repair synthesis by DNA polymerases or sealing by classic DNA ligases. The noncanonical ligase RtcB can guanylylate the DNA 3′-PO₄ to form a DNA_3′pp_5′G_OH cap. Here we show that DNA capping precludes end joining by classic ATP-dependent and NAD⁺-dependent DNA ligases, prevents template-independent nucleotide addition by mammalian terminal transferase, blocks exonucleolytic proofreading by Escherichia coli DNA polymerase II and inhibits proofreading by E. coli DNA polymerase III, while permitting templated DNA synthesis from the cap guanosine 3′-OH primer by E. coli DNA polymerase II (B family) and E. coli DNA polymerase III (C family). Human DNA polymerase β (X family) extends the cap primer predominantly by a single templated addition step. Cap-primed synthesis by templated polymerases embeds a pyrophosphate-linked ribonucleotide in DNA. We find that the embedded ppG is refractory to surveillance and incision by RNase H2.     

Polymerase manager protein UmuD directly regulates Escherichia coli DNA polymerase III α binding to ssDNA

Replication by Escherichia coli DNA polymerase III is disrupted on encountering DNA damage. Consequently, specialized Y-family DNA polymerases are used to bypass DNA damage. The protein UmuD is extensively involved in modulating cellular responses to DNA damage and may play a role in DNA polymerase exchange for damage tolerance. In the absence of DNA, UmuD interacts with the α subunit of DNA polymerase III at two distinct binding sites, one of which is adjacent to the single-stranded DNA-binding site of α. Here, we use single molecule DNA stretching experiments to demonstrate that UmuD specifically inhibits binding of α to ssDNA. We predict using molecular modeling that UmuD residues D91 and G92 are involved in this interaction and demonstrate that mutation of these residues disrupts the interaction. Our results suggest that competition between UmuD and ssDNA for α binding is a new mechanism for polymerase exchange.     

DNA polymerase III chi subunit ties single-stranded DNA binding protein to the bacterial replication machinery

Single-stranded DNA binding (SSB) protein binds to single-stranded DNA (ssDNA) at the lagging strand of the replication fork in Escherichia coli cells. This protein is essential for the survival of the E.coli cell, presumably because it shields the ssDNA and holds it in a suitable conformation for replication by DNA polymerase III. In this study we undertook a biophysical analysis of the interaction between the SSB protein of E.coli and the χ subunit of DNA polymerase III. Using analytical ultracentrifugation we show that at low salt concentrations there is an increase in the stability in the physical interaction between χ and an EcoSSB/ssDNA complex when compared to that of χ to EcoSSB alone. This increase in stability disappeared in high salt conditions. The sedimentation of an EcoSSB protein lacking its C-terminal 26 amino acids remains unchanged in the presence of χ, showing that χ interacts specifically with the C-terminus of EcoSSB. In DNA melting experiments we demonstrate that χ specifically enhances the ssDNA stabilization by EcoSSB. Thus, the binding of EcoSSB to χ at the replication fork prevents premature dissociation of EcoSSB from the lagging strand and thereby enhances the processivity of DNA polymerase III.     

Nicotinic Receptor Subunit α5 Modifies Assembly,Up-regulation,and Response to Pro-inflammatory Cytokines

In the mammalian brain high affinity nicotine-binding sites are composed of at least the α4 and β2 subunits. Additional nicotinic acetylcholine receptor subunits that are often co-expressed with α4+β2 include α5. The introduction of α5 into 293 cells expressing α4+β2 strongly favors assembly of α4+α5+β2 receptors, increases constitutive ligand binding density as measured using [³H]epibatidine, but reduces the magnitude of up-regulation in response to chronic nicotine. In contrast, when β4 is substituted for β2, α5 interferes with the assembly of these receptors, demonstrating an important role for the β subunit in this process. When cells co-express α4+α5+β2+β4, over 50% of the subunit associations include all four subunits, but they fail to be detected using [³H]epibatidine binding. However, complexes of α4+α5+β2 do preferentially emerge from these subunit mixtures, and these mixtures bind ligand. In previous studies of α4+β2+β4 co-expression by 293 cells, the inflammatory cytokines IL-1β and TNFα influenced the outcome of receptor assembly (Gahring, L. C., Days, E. L., Kaasch, T., González de Mendoza, M., Owen, L., Persiyanov, K., and Rogers, S. W. (2005) J. Neuroimmunol. 166, 88–101). When α5 is included in this subunit mixture, and cells are exposed to either inflammatory cytokine, subunit association is no longer altered. These findings suggest that α5 is an influential modulator of α4+β2 nicotinic acetylcholine receptor assembly and stabilizes their expression in response to fluctuations in external conditions.     

Evolutionary clues to DNA polymerase III beta clamp structural mechanisms

The prokaryotic DNA polymerase III β homodimeric clamp links the replication complex to DNA during polynucleotide synthesis. This clamp is loaded onto DNA and unloaded by the clamp loader complex, the δ subunit of which by itself can bind to and open the clamp. β Clamps from diverse bacteria were examined using contrast hierarchical alignment and interaction network (CHAIN) analysis, a statistical approach that categorizes and measures the evolutionary constraints imposed on protein sequences by natural selection. Some constraints are subtle inasmuch as they are unique to certain bacteria. Examination of corresponding molecular interactions within structures of the Escherichia coli β dimeric and δ–β complexes reveals that N320, Y323 and R176, which are subject to very strong constraints, form a substructure that may serve as a platform for leveraging and directing δ-induced conformational changes. N320 may play a prominent role, as it is strategically situated between this substructure and regions linked to δ binding and opening of β’s dimeric interface. R176 appears to act as a relay between the δ binding site and the clamp’s central hole. Other residues subject to strong constraints are likewise associated with structurally important features. For example, two pairs of interacting residues, R269/E304 and K74/E300, form salt bridges at the dimeric interface, while the C-terminal residues M362, P363, M364 and R365 appear to play key roles in δ binding. Q149 and K198 appear to sense DNA within the clamp’s central hole while other residues may relay this information to the δ binding site. Mutagenesis experiments designed to explore possible mechanisms are proposed.     

The C-terminus of Dpb2 is required for interaction with Pol2 and for cell viability

DNA polymerase ε (Pol ε) participates in the synthesis of the leading strand during DNA replication in Saccharomyces cerevisiae. Pol ε comprises four subunits: the catalytic subunit, Pol2, and three accessory subunits, Dpb2, Dpb3 and Dpb4. DPB2 is an essential gene with unclear function. A genetic screen was performed in S. cerevisiae to isolate lethal mutations in DPB2. The dpb2-200 allele carried two mutations within the last 13 codons of the open reading frame, one of which resulted in a six amino acid truncation. This truncated Dpb2 subunit was co-expressed with Pol2, Dpb3 and Dpb4 in S. cerevisiae, but this Dpb2 variant did not co-purify with the other Pol ε subunits. This resulted in the purification of a Pol2/Dpb3/Dpb4 complex that possessed high specific activity and high processivity and holoenzyme assays with PCNA, RFC and RPA on a single-primed circular template did not reveal any defects in replication efficiency. In conclusion, the lack of Dpb2 did not appear to have a negative effect on Pol ε activity. Thus, the C-terminal motif of Dpb2 that we have identified may instead be required for Dpb2 to fulfill an essential structural role at the replication origin or at the replication fork.     

Mapping of the Interaction Between Agrobacterium tumefaciens and Vanda Kasem’s Delight Orchid Protocorm-Like Bodies

Physical contact between A. tumefaciens and the target plant cell walls is essential to transfer and integrate the transgene to introduce a novel trait. Chemotaxis response and attachment of Agrobacterium towards Vanda Kasem’s Delight (VKD) protocorm-like bodies (PLBs) were studied to analyse the interaction between Agrobacterium and PLB during the transformation event. The study shows that initially A. tumefaciens reversibly attached to PLB surface via polar and lateral mode of adherence followed by the irreversible attachment which involved the production of cellulosic fibril by A. tumefaciens. Cellulosic fibril allows formation of biofilm at the tip of trichome. Contrarily, attachment mutant Escherichia coli strain DH5α was significantly deficient in the attachment process. Spectrophotometric GUS assay showed the mean value of attachment by A. tumefaciens was 8.72 % compared to the negative control E. coli strain DH5α that produced 0.16 %. A. tumefaciens swarmed with sharper and brighter edge when severe wounding was applied to the PLBs producing the highest swarming ratio of 1.46 demonstrating the positive effect of the plant exudates on bacterial movement. The study shows that VKD’s PLBs are the suitable explants for Agrobacterium-mediated transformation since the bacteria expressed higher competency rate.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-015-0519-7) contains supplementary material, which is available to authorized users.     

Implication from the predicted docked interaction of sigma H and exploration of its interaction with RNA polymerase in Mycobacterium tuberculosis

M. tuberculosis is adapted to remain active in the extreme environmental condition due to the presence of atypical sigma factors commonly called extra cytoplasmic function (ECF) sigma factors. Among the 13 sigma factors of M. tuberculosis, 10 are regarded as the ECF sigma factor that exerts their attributes in various stress response. Therefore it is of interest to describe the structural prediction of one of the ECF sigma factors, sigma H (SigH), involved in oxidative and heat stress having interaction with the β׳ subunit of M. tuberculosis. RNA polymerase (Mtb-RNAP). The model of Mtb-SigH was build using the commercial package of Discovery Studio version 2.5 from Accelerys (San Diego, CA, USA) containing the inbuilt MODELER module and that of β׳ subunit of Mtb-RNAP using Phyre Server. Further, the protein models were docked using the fully automated web tool ClusPro (cluspro.bu.edu/login.php). Mtb-SigH is a triple helical structure having a putative DNA-binding site and the β׳ subunit of MtbRNAP consists of 18-beta sheets and 22 helices. The SigH-Mtb-RNAP β׳ interaction studies showed that Arg26, Gln19 andAsp18, residues of SigH protein are involved in binding with Arg137, Gln140, Arg152, Asn133 and Asp144 of β׳ subunit of Mtb-RNAP. The predicted model helps to explore the molecular mechanism in the control of gene regulation with a novel unique target for potential new generation inhibitor.     

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can be generated using a transposome. The commercially available, Tn5-derived transposome used in this protocol consists of a linear segment of DNA containing an R6Kγ replication origin, a gene for kanamycin resistance and two mosaic sequence ends, which serve as transposase binding sites. The transposome, provided as a DNA/transposase protein complex, is introduced by electroporation into the prototrophic strain, Enterobacter sp. YSU, and randomly incorporates itself into this host’s genome. Transformants are replica plated onto Luria-Bertani agar plates containing kanamycin, (LB-kan) and onto M-9 medium agar plates containing kanamycin (M-9-kan). The transformants that grow on LB-kan plates but not on M-9-kan plates are considered to be auxotrophs. Purified genomic DNA from an auxotroph is partially digested, ligated and transformed into a pir⁺ Escherichia coli (E. coli) strain. The R6Kγ replication origin allows the plasmid to replicate in pir⁺ E. coli strains, and the kanamycin resistance marker allows for plasmid selection. Each transformant possesses a new plasmid containing the transposon flanked by the interrupted chromosomal region. Sanger sequencing and the Basic Local Alignment Search Tool (BLAST) suggest a putative identity of the interrupted gene. There are three advantages to using this transposome mutagenesis strategy. First, it does not rely on the expression of a transposase gene by the host. Second, the transposome is introduced into the target host by electroporation, rather than by conjugation or by transduction and therefore is more efficient. Third, the R6Kγ replication origin makes it easy to identify the mutated gene which is partially recovered in a recombinant plasmid. This technique can be used to investigate the genes involved in other characteristics of Enterobacter sp. YSU or of a wider variety of bacterial strains.     

Contributions of the individual hydrophobic clefts of the Escherichia coli β sliding clamp to clamp loading, DNA replication and clamp recycling

The homodimeric Escherichia coli β sliding clamp contains two hydrophobic clefts with which proteins involved in DNA replication, repair and damage tolerance interact. Deletion of the C-terminal five residues of β (β^C) disrupted both clefts, severely impairing interactions of the clamp with the DnaX clamp loader, as well as the replicative DNA polymerase, Pol III. In order to determine whether both clefts were required for loading clamp onto DNA, stimulation of Pol III replication and removal of clamp from DNA after replication was complete, we developed a method for purification of heterodimeric clamp proteins comprised of one wild-type subunit (β⁺), and one β^C subunit (β⁺/β^C). The β⁺/β^C heterodimer interacted normally with the DnaX clamp loader, and was loaded onto DNA slightly more efficiently than was β⁺. Moreover, β⁺/β^C interacted normally with Pol III, and stimulated replication to the same extent as did β⁺. Finally, β⁺/β^C was severely impaired for unloading from DNA using either DnaX or the δ subunit of DnaX. Taken together, these findings indicate that a single cleft in the β clamp is sufficient for both loading and stimulation of Pol III replication, but both clefts are required for unloading clamp from DNA after replication is completed.     

Epigenetic silencing of Na,K-ATPase β1 subunit gene ATP1B1 by methylation in clear cell renal cell carcinoma

The Na,K-ATPase or sodium pump carries out the coupled extrusion of Na⁺ and uptake of K⁺ across the plasma membranes of cells of most higher eukaryotes. We have shown earlier that Na,K-ATPase-β₁ (NaK-β) protein levels are highly reduced in poorly differentiated kidney carcinoma cells in culture and in patients' tumor samples. The mechanism(s) regulating the expression of NaK-β in tumor tissues has yet to be explored. We hypothesized that DNA methylation plays a role in silencing the NaK-β gene (ATP1B1) expression in kidney cancers. In this study, to the best of our knowledge we provide the first evidence that ATP1B1 is epigenetically silenced by promoter methylation in both renal cell carcinoma (RCC) patients’ tissues and cell lines. We also show that knockdown of the von Hippel-Lindau (VHL) tumor suppressor gene in RCC cell lines results in enhanced ATP1B1 promoter AT hypermethylation, which is accompanied by reduced expression of NaK-β. Furthermore, treatment with 5-Aza-2′-deoxycytidine rescued the expression of ATP1B1 mRNA as well as NaK-β protein in these cells. These data demonstrate that promoter hypermethylation is associated with reduced NaK-β expression, which might contribute to RCC initiation and/or disease progression.     

Rice transgenic plants with suppressed expression of the β subunit of the heterotrimeric G protein

The deficient mutant for the rice heterotrimeric G protein α subunit gene (RGA1), d1, showed dwarfism and set small seed due to a reduced cell number. Mutants for the rice heterotrimeric G protein β subunit gene (RGB1) have not been isolated. To determine the functions of RGB1, transgenic rice plants with suppressed expression of RGB1 were studied using the RNAi method. RGB1 knock-down lines showed browning of the lamina joint regions and nodes and reduced fertility, but these abnormality were not observed in d1. Transgenic plants in which the G protein β subunit was greatly decreased were not obtained, suggesting that the complete suppression of RGB1 mRNA may be lethal. In contrast, the d1 mutants, with complete loss of the G protein α subunit, were fertile and half the size of the WT. These studies suggest that RGB1 has different functions than RGA1.     

The Human Lagging Strand DNA Polymerase δ Holoenzyme Is Distributive

Polymerase δ is widely accepted as the lagging strand replicative DNA polymerase in eukaryotic cells. It forms a replication complex in the presence of replication factor C and proliferating cell nuclear antigen to perform efficient DNA synthesis in vivo. In this study, the human lagging strand holoenzyme was reconstituted in vitro. The rate of DNA synthesis of this holoenzyme, measured with a singly primed ssM13 DNA substrate, is 4.0 ± 0.4 nucleotides. Results from adenosine 5′-(3-thiotriphosphate) tetralithium salt (ATPγS) inhibition experiments revealed the nonprocessive characteristic of the human DNA polymerase (Pol δ) holoenzyme (150 bp for one binding event), consistent with data from chase experiments with catalytically inactive mutant Pol δ^AA. The ATPase activity of replication factor C was characterized and found to be stimulated ∼10-fold in the presence of both proliferating cell nuclear antigen and DNA, but the activity was not shut down by Pol δ in accord with rapid association/dissociation of the holoenzyme to/from DNA. It is noted that high concentrations of ATP inhibit the holoenzyme DNA synthesis activity, most likely due to its inhibition of the clamp loading process.