Isolation and characterization of a second subunit of molecular chaperonin from Pyrococcus kodakaraensis KOD1: analysis of an ATPase-deficient mutant enzyme

The cpkA gene encoding a second (alpha) subunit of archaeal chaperonin from Pyrococcus kodakaraensis KOD1 was cloned, sequenced, and expressed in Escherichia coli. Recombinant CpkA was studied for chaperonin functions in comparison with CpkB (beta subunit). The effect on decreasing the insoluble form of proteins was examined by coexpressing CpkA or CpkB with CobQ (cobyric acid synthase from P. kodakaraensis) in E. coli. The results indicate that both CpkA and CpkB effectively decrease the amount of the insoluble form of CobQ. Both CpkA and CpkB possessed the same ATPase activity as other bacterial and eukaryal chaperonins. The ATPase-deficient mutant proteins CpkA-D95K and CpkB-D95K were constructed by changing conserved Asp95 to Lys. Effect of the mutation on the ATPase activity and CobQ solubilization was examined. Neither mutant exhibited ATPase activity in vitro. Nevertheless, they decreased the amount of the insoluble form of CobQ by coexpression as did wild-type CpkA and CpkB. These results implied that both CpkA and CpkB could assist protein folding for nascent protein in E. coli without requiring energy from ATP hydrolysis.     

In vitro stabilization and in vivo solubilization of foreign proteins by the beta subunit of a chaperonin from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1.

The gene encoding the beta subunit of a molecular chaperonin from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1 (cpkB) was cloned, sequenced, and expressed in Escherichia coli. The cpkB gene is composed of 1,641 nucleotides, encoding a protein (546 amino acids) with a molecular mass of 59,140 Da. The enhancing effect of CpkB on enzyme stability was examined by using Saccharomyces cerevisiae alcohol dehydrogenase (ADH). Purified recombinant CpkB prevents thermal denaturation and enhances thermostability of ADH. CpkB requires ATP for its chaperonin function at a low CpkB concentration; however, CpkB functions without ATP when present in excess. In vivo chaperonin function for the solubilization of insoluble proteins was also studied by coexpressing CpkB and CobQ (cobryic acid synthase), indicating that CpkB is useful for solubilizing the insoluble proteins in vivo. These results suggest that the beta subunit plays a major role in chaperonin activity and is functional without the alpha subunit.     

Two kinds of archaeal chaperonin with different temperature dependency from a hyperthermophile

Thermococcus kodakaraensis KOD1 produces two kinds of chaperonin subunits, CpkA and CpkB. To monitor the expression levels of CpkA and CpkB, anti-CpkA and anti-CpkB antisera were obtained by using synthesized peptides as the haptens. These haptens were prepared based on the carboxyl terminus regions of CpkA and CpkB, which show clear differences in amino acid sequence. Immunoblotting analysis using obtained antisera revealed that the expression levels of CpkA and CpkB changed depending on the cultivation temperature. When cells were grown at 95 degrees C, intracellular amount of CpkA was low, while CpkB was expressed at extremely high level in KOD1. In the case of 70 degrees C cultivation, CpkA existed as the major chaperonin in the cell, whereas CpkB existed as the minor one. Temperature-shift experiments showed that the expression of CpkB was induced by the up-shift and reduced by the down-shift of temperature. In contrast, the expression of CpkA was reduced by the up-shift and induced by the down-shift of temperature. Furthermore, native PAGE and immunoprecipitation experiments revealed that the stoichiometrical ratio of CpkA and CpkB in chaperonin complex changed according to growth temperature.     

Temperature-Sensitive Modification and Restriction Phenotypes of an Escherichia coli dnaD Mutant

A mutant of Escherichia coli temperature-sensitive for deoxyribonucleic acid synthesis, dnaD, was found to have temperature-sensitive modification and restriction phenotypes. In contrast to the original observation by Carl (1970), the mutant could support the growth of λ phage at 41 C. However, the λ phages thus produced were able to form plaques with normal plating efficiency only on E. coli C, a restriction-less strain, but not on E. coli K. Since the λ phages produced in the mutant at 30 C could form plaques equally well on both E. coli strains, it was concluded that the dnaD mutant has a temperature-sensitive modification phenotype. Furthermore, since the dnaD mutant allowed some growth of unmodified λ·C phages at 41 C but less at 30 C, the mutant is also temperature sensitive in restriction. The relationship, if any, between temperature-sensitive deoxyribonucleic acid synthesis and temperature-sensitive modification-restriction in the dnaD mutant is not known. Similar experiments were done with three dnaC mutants and one dnaA mutant. Two dnaC mutants were found to have altered restriction phenotypes at 41 C, but none of the mutants were defective in modification.     

Ca2+-Dependent Maturation of Subtilisin from a Hyperthermophilic Archaeon, Thermococcus kodakaraensis: the Propeptide Is a Potent Inhibitor of the Mature Domain but Is Not Required for Its Folding

Subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 is a member of the subtilisin family. T. kodakaraensis subtilisin in a proform (T. kodakaraensis pro-subtilisin), as well as its propeptide (T. kodakaraensis propeptide) and mature domain (T. kodakaraensis mat-subtilisin), were independently overproduced in E. coli, purified, and biochemically characterized. T. kodakaraensis pro-subtilisin was inactive in the absence of Ca²⁺ but was activated upon autoprocessing and degradation of propeptide in the presence of Ca²⁺ at 80°C. This maturation process was completed within 30 min at 80°C but was bound at an intermediate stage, in which the propeptide is autoprocessed from the mature domain (T. kodakaraensis mat-subtilisin*) but forms an inactive complex with T. kodakaraensis mat-subtilisin*, at lower temperatures. At 80°C, approximately 30% of T. kodakaraensis pro-subtilisin was autoprocessed into T. kodakaraensis propeptide and T. kodakaraensis mat-subtilisin*, and the other 70% was completely degraded to small fragments. Likewise, T. kodakaraensis mat-subtilisin was inactive in the absence of Ca²⁺ but was activated upon incubation with Ca²⁺ at 80°C. The kinetic parameters and stability of the resultant activated protein were nearly identical to those of T. kodakaraensis mat-subtilisin*, indicating that T. kodakaraensis mat-subtilisin does not require T. kodakaraensis propeptide for folding. However, only ~5% of T. kodakaraensis mat-subtilisin was converted to an active form, and the other part was completely degraded to small fragments. T. kodakaraensis propeptide was shown to be a potent inhibitor of T. kodakaraensis mat-subtilisin* and noncompetitively inhibited its activity with a K_i of 25 ± 3.0 nM at 20°C. T. kodakaraensis propeptide may be required to prevent the degradation of the T. kodakaraensis mat-subtilisin molecules that are activated later by those that are activated earlier.     

Affinity labeling of purified Ca2+,Mg2+-activated ATPase of Escherichia coli by the 2',3'-dialdehydes of adenosine 5'-di- and triphosphates

The 2′,3′-dialdehydes of ADP and ATP (oADP and oATP), obtained by periodate oxidation of ADP and ATP, inhibited the hydrolytic activity of the purified Ca²⁺.Mg²⁺-activated ATPase of Escherichia coli. Nonspecific labeling of amino groups by these dialdehydes was corrected by carrying out the reactions in the presence of 15 mm ATP. Two types of modification of “ATP-protectable” binding sites by oATP could be detected. The binding of 2 mol “ATP-protectable” oATP/mol ATPase was without affect on ATPase activity and still occurred in the hydrolytically inactive ATPase of an unc A mutant. The binding of a further 3 mol “ATP-protectable” oATP/mol ATPase resulted in almost complete loss of ATPase activity although much of the loss occurred during the binding of the first additional molecule of oATP. This additional ATP-protectable oATP binding did not occur in the unc A mutant and so resembled both the inhibitory effect of oADP on the ATPase activity of normal strains and its lack of binding to the unc A ATPase (P. D. Bragg and C. Hou, 1980, Biochem. Biophys. Res. Commun.95, 952–957). The “ATP-protectable” binding sites for oADP and oATP were located on the α subunit of the ATPase. Binding of oADP or oATP did not result in release of the tightly bound ADP and ATP from the enzyme. We conclude that separate binding sites for oADP and oATP occur on the α subunits of the E. coli ATPase and that the former may be the active site(s) for ATP hydrolysis while the latter are involved in regulation of the ATPase complex.     

Human CCT4 and CCT5 Chaperonin Subunits Expressed in Escherichia coli Form Biologically Active Homo-oligomers

Chaperonins are a family of chaperones that encapsulate their substrates and assist their folding in an ATP-dependent manner. The ubiquitous eukaryotic chaperonin, TCP-1 ring complex (TRiC), is a hetero-oligomeric complex composed of two rings, each formed from eight different CCT (chaperonin containing TCP-1) subunits. Each CCT subunit may have distinct substrate recognition and ATP hydrolysis properties. We have expressed each human CCT subunit individually in Escherichia coli to investigate whether they form chaperonin-like double ring complexes. CCT4 and CCT5, but not the other six CCT subunits, formed high molecular weight complexes within the E. coli cells that sedimented about 20S in sucrose gradients. When CCT4 and CCT5 were purified, they were both organized as two back-to-back rings of eight subunits each, as seen by negative stain and cryo-electron microscopy. This morphology is consistent with that of the hetero-oligomeric double-ring TRiC purified from bovine testes and HeLa cells. Both CCT4 and CCT5 homo-oligomers hydrolyzed ATP at a rate similar to human TRiC and were active as assayed by luciferase refolding and human γD-crystallin aggregation suppression and refolding. Thus, both CCT4 and CCT5 homo-oligomers have the property of forming 8-fold double rings absent the other subunits, and these complexes carry out chaperonin reactions without other partner subunits.     

Reconstitution of ATPase from the isolated subunits of coupling factor F1's of Escherichia coli and thermophilic bacterium PS3

ATPase was reconstituted from mixtures of isolated subunits of coupling factor, F₁ ATPase of E. coli (EF₁) and thermophilic bacterium PS3 (TF₁); ability to hydrolyze ATP was attained from the combination of α and β subunits from EF₁ and γ subunit from TF₁, α and β from TF₁ and γ from EF₁, and α and γ from EF₁ and β from TF₁. The β subunit of TF₁ also could complement the EF₁ from an E. coli mutant defective in this subunit. This is the first demonstration of interspecies in vitro recombination of ATPase activity from isolated subunits.     

Functional characterization of recombinant prefoldin complexes from a hyperthermophilic archaeon, Thermococcus sp. strain KS-1

Prefoldin is a heterohexameric molecular chaperone complex that is found in the eukaryotic cytosol and also in archaea. It captures a nonnative protein and subsequently delivers it to a group II chaperonin for proper folding. Archaeal prefoldin is a heterocomplex containing two α subunits and four β subunits with the structure of a double β-barrel assembly, with six long coiled coils protruding from it like a jellyfish with six tentacles. We have studied the protein folding mechanism of group II chaperonin using those of Thermococcus sp. strain KS-1 (T. KS-1) because they exhibit high protein folding activity in vitro. We have also demonstrated functional cooperation between T. KS-1 chaperonins and prefoldin from Pyrococcus horikoshii OT3. Recent genome analysis has shown that Thermococcus kodakaraensis KOD1 contains two pairs of prefoldin subunit genes, correlating with the existence of two different chaperonin subunits. In this study, we characterized four different recombinant prefoldin complexes composed of two pairs of prefoldin subunits (α1, α2, β1, and β2) from T. KS-1. All of them (α1-β1, α2-β1, α1-β2, and α2-β2) exist as α₂β₄ heterohexamers and can protect several proteins from forming aggregates with different activities. We have also compared the collaborative activity between the prefoldin complexes and the cognate chaperonins. Prefoldin complexes containing the β1 subunit interacted with the chaperonins more strongly than those with the β2 subunit. The results suggest that Thermococcus spp. express different prefoldins for different substrates or conditions as chaperonins.     

Mutant DnaAs of Escherichia coli that are refractory to negative control

DnaA is the initiator of DNA replication in bacteria. A mutant DnaA named DnaAcos is unusual because it is refractory to negative regulation. We developed a genetic method to isolate other mutant DnaAs that circumvent regulation to extend our understanding of mechanisms that control replication initiation. Like DnaAcos, one mutant bearing a tyrosine substitution for histidine 202 (H202Y) withstands the regulation exerted by datA, hda and dnaN (β clamp), and both DnaAcos and H202Y resist inhibition by the Hda-β clamp complex in vitro. Other mutant DnaAs carrying G79D, E244K, V303M or E445K substitutions are either only partially sensitive or refractory to inhibition by the Hda-β clamp complex in vitro but are responsive to hda expression in vivo. All mutant DnaAs remain able to interact directly with Hda. Of interest, both DnaAcos and DnaAE244K bind more avidly to Hda. These mutants, by sequestrating Hda, may limit its availability to regulate other DnaA molecules, which remain active to induce extra rounds of DNA replication. Other evidence suggests that a mutant bearing a V292M substitution hyperinitiates by escaping the effect of an unknown regulatory factor. Together, our results provide new insight into the mechanisms that regulate replication initiation in Escherichia coli.     

Characterisation of the DNA-dependent ATPase activity of human DNA topoisomerase IIbeta: mutation of Ser165 in the ATPase domain reduces the ATPase activity and abolishes the in vivo complementation ability

We report for the first time an analysis of the ATPase activity of human DNA topoisomerase (topo) IIβ. We show that topo IIβ is a DNA-dependent ATPase that appears to fit Michaelis–Menten kinetics. The ATPase activity is stimulated 44-fold by DNA. The k_cat for ATP hydrolysis by human DNA topo IIβ in the presence of DNA is 2.25 s^–1. We have characterised a topo IIβ derivative which carries a mutation in the ATPase domain (S165R). S165R reduced the k_cat for ATP hydrolysis by 7-fold, to 0.32 s^–1, while not significantly altering the apparent K_m. The specificity constant for the interaction between ATP and topo IIβ (k_cat/K_mapp) showed a 90% reduction for βS165R. The DNA binding affinity and ATP-independent DNA cleavage activity of the enzyme are unaffected by this mutation. However, the strand passage activity is reduced by 80%, presumably due to reduced ATP hydrolysis. The mutant enzyme is unable to complement ts yeast topo II in vivo. We have used computer modelling to predict the arrangement of key residues at the ATPase active site of topo IIβ. Ser165 is predicted to lie very close to the bound nucleotide, and the S165R mutation could thus influence both ATP binding and ADP dissociation.     

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.     

The Torque of Rotary F-ATPase Can Unfold Subunit Gamma If Rotor and Stator Are Cross-Linked

During ATP hydrolysis by F₁-ATPase subunit γ rotates in a hydrophobic bearing, formed by the N-terminal ends of the stator subunits (αβ)₃. If the penultimate residue at the α-helical C-terminal end of subunit γ is artificially cross-linked (via an engineered disulfide bridge) with the bearing, the rotary function of F₁ persists. This observation has been tentatively interpreted by the unfolding of the α-helix and swiveling rotation in some dihedral angles between lower residues. Here, we screened the domain between rotor and bearing where an artificial disulfide bridge did not impair the rotary ATPase activity. We newly engineered three mutants with double cysteines farther away from the C-terminus of subunit γ, while the results of three further mutants were published before. We found ATPase and rotary activity for mutants with cross-links in the single α-helical, C-terminal portion of subunit γ (from γ285 to γ276 in E. coli), and virtually no activity when the cross-link was placed farther down, where the C-terminal α-helix meets its N-terminal counterpart to form a supposedly stable coiled coil. In conclusion, only the C-terminal singular α-helix is prone to unwinding and can form a swivel joint, whereas the coiled coil portion seems to resist the enzyme''s torque.     

Probing the functional mechanism of Escherichia coli GroEL using circular permutation

Background

The Escherichia coli chaperonin GroEL subunit consists of three domains linked via two hinge regions, and each domain is responsible for a specific role in the functional mechanism. Here, we have used circular permutation to study the structural and functional characteristics of the GroEL subunit.

Methodology/Principal Findings

Three soluble, partially active mutants with polypeptide ends relocated into various positions of the apical domain of GroEL were isolated and studied. The basic functional hallmarks of GroEL (ATPase and chaperoning activities) were retained in all three mutants. Certain functional characteristics, such as basal ATPase activity and ATPase inhibition by the cochaperonin GroES, differed in the mutants while at the same time, the ability to facilitate the refolding of rhodanese was roughly equal. Stopped-flow fluorescence experiments using a fluorescent variant of the circularly permuted GroEL CP376 revealed that a specific kinetic transition that reflects movements of the apical domain was missing in this mutant. This mutant also displayed several characteristics that suggested that the apical domains were behaving in an uncoordinated fashion.

Conclusions/Significance

The loss of apical domain coordination and a concomitant decrease in functional ability highlights the importance of certain conformational signals that are relayed through domain interlinks in GroEL. We propose that circular permutation is a very versatile tool to probe chaperonin structure and function.     

Inhibition of F1-ATPase Rotational Catalysis by the Carboxyl-terminal Domain of the ? Subunit

Escherichia coli ATP synthase (F₀F₁) couples catalysis and proton transport through subunit rotation. The ϵ subunit, an endogenous inhibitor, lowers F₁-ATPase activity by decreasing the rotation speed and extending the duration of the inhibited state (Sekiya, M., Hosokawa, H., Nakanishi-Matsui, M., Al-Shawi, M. K., Nakamoto, R. K., and Futai, M. (2010) Single molecule behavior of inhibited and active states of Escherichia coli ATP synthase F₁ rotation. J. Biol. Chem. 285, 42058–42067). In this study, we constructed a series of ϵ subunits truncated successively from the carboxyl-terminal domain (helix 1/loop 2/helix 2) and examined their effects on rotational catalysis (ATPase activity, average rotation rate, and duration of inhibited state). As expected, the ϵ subunit lacking helix 2 caused about ½-fold reduced inhibition, and that without loop 2/helix 2 or helix 1/loop 2/helix 2 showed a further reduced effect. Substitution of ϵSer¹⁰⁸ in loop 2 and ϵTyr¹¹⁴ in helix 2, which possibly interact with the β and γ subunits, respectively, decreased the inhibitory effect. These results suggest that the carboxyl-terminal domain of the ϵ subunit plays a pivotal role in the inhibition of F₁ rotation through interaction with other subunits.     

Active Subtilisin-Like Protease from a Hyperthermophilic Archaeon in a Form with a Putative Prosequence

The gene encoding subtilisin-like protease T. kodakaraensis subtilisin was cloned from a hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. T. kodakaraensis subtilisin is a member of the subtilisin family and composed of 422 amino acid residues with a molecular weight of 43,783. It consists of a putative presequence, prosequence, and catalytic domain. Like bacterial subtilisins, T. kodakaraensis subtilisin was overproduced in Escherichia coli in a form with a putative prosequence in inclusion bodies, solubilized in the presence of 8 M urea, and refolded and converted to an active molecule. However, unlike bacterial subtilisins, in which the prosequence was removed from the catalytic domain by autoprocessing upon refolding, T. kodakaraensis subtilisin was refolded in a form with a putative prosequence. This refolded protein of recombinant T. kodakaraensis subtilisin which is composed of 398 amino acid residues (Gly⁻⁸² to Gly³¹⁶), was purified to give a single band on a sodium dodecyl sulfate (SDS)-polyacrylamide gel and characterized for biochemical and enzymatic properties. The good agreement of the molecular weights estimated by SDS-polyacrylamide gel electrophoresis (44,000) and gel filtration (40,000) suggests that T. kodakaraensis subtilisin exists in a monomeric form. T. kodakaraensis subtilisin hydrolyzed the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide only in the presence of the Ca²⁺ ion with an optimal pH and temperature of pH 9.5 and 80°C. Like bacterial subtilisins, it showed a broad substrate specificity, with a preference for aromatic or large nonpolar P1 substrate residues. However, it was much more stable than bacterial subtilisins against heat inactivation and lost activity with half-lives of >60 min at 80°C, 20 min at 90°C, and 7 min at 100°C.     

Asp-52 in Combination with Asp-398 Plays a Critical Role in ATP Hydrolysis of Chaperonin GroEL

The Escherichia coli chaperonin GroEL is a double-ring chaperone that assists protein folding with the aid of GroES and ATP. Asp-398 in GroEL is known as one of the critical residues on ATP hydrolysis because GroEL(D398A) mutant is deficient in ATP hydrolysis (<2% of the wild type) but not in ATP binding. In the archaeal Group II chaperonin, another aspartate residue, Asp-52 in the corresponding E. coli GroEL, in addition to Asp-398 is also important for ATP hydrolysis. We investigated the role of Asp-52 in GroEL and found that ATPase activity of GroEL(D52A) and GroEL(D52A/D398A) mutants was ∼20% and <0.01% of wild-type GroEL, respectively, indicating that Asp-52 in E. coli GroEL is also involved in the ATP hydrolysis. GroEL(D52A/D398A) formed a symmetric football-shaped GroEL-GroES complex in the presence of ATP, again confirming the importance of the symmetric complex during the GroEL ATPase cycle. Notably, the symmetric complex of GroEL(D52A/D398A) was extremely stable, with a half-time of ∼150 h (∼6 days), providing a good model to characterize the football-shaped complex.     

Indole-3-glycerol-phosphate synthase is recognized by a cold-inducible group II chaperonin in Thermococcus kodakarensis

Thermococcus kodakarensis optimally grows at 85°C and possesses two chaperonins, cold-inducible CpkA and heat-inducible CpkB. Gene disruptants DA1 (ΔcpkA) and DB1 (ΔcpkB) showed decreased cell growth at 60°C and 93°C, respectively. The DB2 mutant (ΔcpkAcpkB ΔcpkB), whose cpkB gene was expressed under the control of the cpkA promoter, did not grow at 60°C, and the DB3 mutant [ΔcpkA(1-524)cpkB(1-524) ΔcpkB], whose CpkA amino acid residues 1 to 524 were replaced with corresponding CpkB residues that maintained the C-terminal region intact, grew at 60°C, implying that the CpkA C-terminal region plays a key role in cell growth at 60°C. To screen for specific CpkA target proteins, comparative pulldown studies with anti-Cpk were performed using cytoplasmic fractions from DA1 cells cultivated at 93°C and DB1 cells cultivated at 60°C. Among the proteins coprecipitated with anti-Cpk, TK0252, encoding indole-3-glycerol-phosphate synthase (TrpC), showed the highest Mascot score. Counter-pulldown experiments were also performed on DA1 and DB1 extracts using anti-TrpC. CpkA coimmunoprecipitated with anti-TrpC while CpkB did not. The results obtained indicate that TrpC is a specific target for CpkA. The effects of Cpks on denatured TrpC were then examined. The refolding of partially denatured TrpC was accelerated by the addition of CpkA but not by adding CpkB. DA1 cells grew optimally in minimal medium only in the presence of tryptophan but hardly grew in the absence of tryptophan at 60°C. It has been suggested that a lesion of functional TrpC is caused by cpkA disruption, resulting in tryptophan auxotrophy.     

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III ε subunit (featuring 3’-5’ exonuclease activity) is highly improved when the same protein is co-expressed with the α and θ subunits (featuring DNA polymerase activity and stabilizing ε, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the α subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the ε subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type ε subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-β-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.     

Escherichia coli HU protein has a role in the repair of abasic sites in DNA

HU is one of the most abundant DNA binding proteins in Escherichia coli. We find that it binds strongly to DNA containing an abasic (AP) site or tetrahydrofuran (THF) (apparent K_d ≈50 nM). It also possesses an AP lyase activity that cleaves at a deoxyribose but not at a THF residue. The binding and cleavage of an AP site was observed only with the HUαβ heterodimer. Site-specific mutations at K3 and R61 residues led to a change in substrate binding and cleavage. Both K3A(α)K3A(β) and R61A(α)R61A(β) mutant HU showed significant reduction in binding to DNA containing AP site; however, only R61A(α)R61A(β) mutant protein exhibited significant loss in AP lyase activity. Both K3A(α)K3A(β) and R61K(α)R61K(β) showed slight reduction in AP lyase activities. The function of HU protein as an AP lyase was confirmed by the ability of hupA or hupB mutations to further reduce the viability of an E. coli dut(Ts) xth mutant, which generates lethal AP sites at 37°C; the hupA and hupB derivatives, respectively, had a 6-fold and a 150-fold lower survival at 37°C than did the parental strain. These data suggest, therefore, that HU protein plays a significant role in the repair of AP sites in E. coli.