Octameric Form Research Articles

Structural and functional analysis of hyper-thermostable ancestral L-amino acid oxidase that can convert Trp derivatives to D-forms by chemoenzymatic reaction

Production of D-amino acids (D-AAs) on a large-scale enables to provide precursors of peptide therapeutics. In this study, we designed a novel L-amino acid oxidase, HTAncLAAO2, by ancestral sequence reconstruction, exhibiting high thermostability and long-term stability. The crystal structure of HTAncLAAO2 was determined at 2.2 Å by X-ray crystallography, revealing that the enzyme has an octameric form like a “ninja-star” feature. Enzymatic property analysis demonstrated that HTAncLAAO2 exhibits three-order larger kcat/Km values towards four L-AAs (L-Phe, L-Leu, L-Met, and L-Ile) than that of L-Trp. Through screening the variants, we obtained the HTAncLAAO2(W220A) variant, which shows a > 6-fold increase in kcat value toward L-Trp compared to the original enzyme. This variant applies to synthesizing enantio-pure D-Trp derivatives from L- or rac-forms at a preparative scale. Given its excellent properties, HTAncLAAO2 would be a starting point for designing novel oxidases with high activity toward various amines and AAs.

Structural Analysis of an Octameric Resorcinarene Self-Assembly in Toluene and its Morphological Transition by Temperature.

Calix[4]resorcinarene derivatives such as C-undecylcalix[4]resorcinarene self-assemble into a hexameric capsule-like structure. This structure is expected in any apolar solvent, but we recently found a self-assembled octameric form of resorcinarene in toluene. To understand this unexpected form, we performed small-angle X-ray scattering (SAXS) measurements of the octameric self-assembly. In ab initio shape reconstruction and model fitting of the SAXS profile, a core-shell spherical structure resembling a reverse micelle was found in the octameric self-assembly. The shell and core were composed of alkyl chains and resorcinol moieties, respectively. We also evaluated the temperature dependence of the octameric self-assembly. Above 95 °C, the structure began directly decomposing into unimers, implying the partial cleavage of hydrogen bonds in the octamer core. Meanwhile, below 25 °C, the spherical structure of the octamer partially transformed to a cylindrical structure. This morphological transition could be understood by packing parameter theory, which is often applied to suspected micellar structures.

Cryo-EM structure of the Rous sarcoma virus octameric cleaved synaptic complex intasome

Despite conserved catalytic integration mechanisms, retroviral intasomes composed of integrase (IN) and viral DNA possess diverse structures with variable numbers of IN subunits. To investigate intasome assembly mechanisms, we employed the Rous sarcoma virus (RSV) IN dimer that assembles a precursor tetrameric structure in transit to the mature octameric intasome. We determined the structure of RSV octameric intasome stabilized by a HIV-1 IN strand transfer inhibitor using single particle cryo-electron microscopy. The structure revealed significant flexibility of the two non-catalytic distal IN dimers along with previously unrecognized movement of the conserved intasome core, suggesting ordered conformational transitions between intermediates that may be important to capture the target DNA. Single amino acid substitutions within the IN C-terminal domain affected intasome assembly and function in vitro and infectivity of pseudotyped RSV virions. Unexpectedly, 17 C-terminal amino acids of IN were dispensable for virus infection despite regulating the transition of the tetrameric intasome to the octameric form in vitro. We speculate that this region may regulate the binding of highly flexible distal IN dimers to the intasome core to form the octameric complex. Our studies reveal key steps in the assembly of RSV intasomes.

The Molecular Mechanism of Domain Swapping of the C-Terminal Domain of the SARS-Coronavirus Main Protease

In three-dimensional domain swapping, two protein monomers exchange a part of their structures to form an intertwined homodimer, whose subunits resemble the monomer. Several viral proteins domain swap to increase their structural complexity or functional avidity. The main protease (Mpro) of the severe acute respiratory syndrome (SARS) coronavirus proteolyzes viral polyproteins and has been a target for anti-SARS drug design. Domain swapping in the α-helical C-terminal domain of Mpro (MproC) locks Mpro into a hyperactive octameric form that is hypothesized to promote the early stages of viral replication. However, in the absence of a complete molecular understanding of the mechanism of domain swapping, investigations into the biological relevance of this octameric Mpro have stalled. Isolated MproC can exist as a monomer or a domain-swapped dimer. Here, we investigate the mechanism of domain swapping of MproC using coarse-grained structure-based models and molecular dynamics simulations. Our simulations recapitulate several experimental features of MproC folding. Further, we find that a contact between a tryptophan in the MproC domain-swapping hinge and an arginine elsewhere forms early during folding, modulates the folding route, and promotes domain swapping to the native structure. An examination of the sequence and the structure of the tryptophan containing hinge loop shows that it has a propensity to form multiple secondary structures and contacts, indicating that it could be stabilized into either the monomer- or dimer-promoting conformations by mutations or ligand binding. Finally, because all residues in the tryptophan loop are identical in SARS-CoV and SARS-CoV-2, mutations that modulate domain swapping may provide insights into the role of octameric Mpro in the early-stage viral replication of both viruses.

Characterization of theCorynebacterium glutamicumdehydroshikimate dehydratase QsuB and its potential for microbial production of protocatechuic acid.

The dehydroshikimate dehydratase (DSD) from Corynebacterium glutamicum encoded by the qsuB gene is related to the previously described QuiC1 protein (39.9% identity) from Pseudomonas putida. Both QuiC1 and QsuB are two-domain bacterial DSDs. The N-terminal domain provides dehydratase activity, while the C-terminal domain has sequence identity with 4-hydroxyphenylpyruvate dioxygenase. Here, the QsuB protein and its N-terminal domain (N-QsuB) were expressed in the T7 system, purified and characterized. QsuB was present mainly in octameric form (60%), while N-QsuB had a predominantly monomeric structure (80%) in aqueous buffer. Both proteins possessed DSD activity with one of the following cofactors (listed in the order of decreasing activity): Co2+, Mg2+, Mn2+. The Km and kcat values for the QsuB enzyme (Km ~ 1 mM, kcat ~ 61 s-1) were two and three times higher than those for N-QsuB. 3,4-DHBA inhibited QsuB (Ki ~ 0.38 mM, Ki’ ~ 0.96 mM) and N-QsuB (Ki ~ 0.69 mM) enzymes via mixed and noncompetitive inhibition mechanism, respectively. E. coli MG1655ΔaroEPlac‒qsuB strain produced three times more 3,4-DHBA from glucose in test tube fermentation than the MG1655ΔaroEPlac‒n-qsuB strain. The C-terminal domain activity towards 3,4-DHBA was not established in vitro. This domain was proposed to promote protein oligomerization for maintaining structural stability of the enzyme. The dimer formation of QsuB protein was more predictable (ΔG = ‒15.8 kcal/mol) than the dimerization of its truncated version N-QsuB (ΔG = ‒0.4 kcal/mol).

Exploring the Nature of Cationic Blocker Recognition by the Anthrax Toxin Channel

Obstructing conductive pathways of the channel-forming toxins with targeted blockers is a promising drug design approach. Nearly all tested positively charged ligands have been shown to reversibly block the cation-selective channel-forming protective antigen (PA63) component of the binary anthrax toxin. The cationic ligands with more hydrophobic surfaces, particularly those carrying aromatic moieties, inhibited PA63 more effectively. To understand the physical basis of PA63 selectivity for a particular ligand, detailed information is required on how the blocker structural elements (e.g., positively charged and aromatic groups) influence the molecular kinetics of the blocker/channel binding reactions. In this study, we address this problem using the high-resolution single-channel planar lipid bilayer technique. Several structurally distinct cationic blockers, namely per-6-S-(3-amino) propyl-β-cyclodextrin, per-6-S-(3-aminomethyl) benzyl-α-cyclodextrin, per-6-S-(3-aminomethyl) benzyl-β-cyclodextrin, per-6-S-(3-aminomethyl) benzyl-γ-cyclodextrin, methyltriphenylphosphonium ion, and G0 polyamidoamine dendrimer are tested for their ability to inhibit the heptameric and octameric PA63 variants and PA63F427A mutant. The F427 residues form a hydrophobic constriction region inside the channel, known as the “ϕ-clamp.” We show that the cationic blockers interact with PA63 through a combination of forces. Analysis of the binding reaction kinetics suggests the involvement of cation-π, Coulomb, and salt-concentration-independent π-π or hydrophobic interactions in the cationic cyclodextrin binding. It is possible that these blockers bind to the ϕ-clamp and are also stabilized by the Coulomb interactions of their terminal amino groups with the water-exposed negatively charged channel residues. In PA63F427A, only the suggested Coulomb component of the cyclodextrin interaction remains. Methyltriphenylphosphonium ion and G0 polyamidoamine dendrimer, despite being positively charged, interact primarily with the ϕ-clamp. We also show that seven- and eightfold symmetric cyclodextrins effectively block the heptameric and octameric forms of PA63 interchangeably, adding flexibility to the earlier formulated blocker/target symmetry match requirement.

The influence of truncating the carboxy-terminal amino acid residues of streptococcal enolase on its ability to interact with canine plasminogen.

The native octameric structure of streptococcal enolase from Streptococcus pyogenes increasingly dissociates as amino acid residues are removed one by one from the carboxy-terminus. These truncations gradually convert native octameric enolase into monomers and oligomers. In this work, we investigated how these truncations influence the interaction between Streptococcal enolase and canine plasminogen. We used dual polarization interferometry (DPI), localized surface plasmon resonance (LSPR), and sedimentation velocity analytical ultracentrifugation (AUC) to study the interaction. The DPI was our first technique, was performed on all the truncations and used one exclusive kind of chip. The LSRP was used to show that the DPI results were not dependent on the type of chip used. The AUC was required to show that our surface results were not the result of selecting a minority population in any given sample; the majority of the protein was responsible for the binding phenomenon we observed. By comparing results from these techniques we identified one detail that is essential for streptococcal enolase to bind plasminogen: In our hands the individual monomers bind plasminogen; dimers, trimers, tetramers may or may not bind, the fully intact, native, octamer does not bind plasminogen. We also evaluated the contribution to the equilibrium constant made by surface binding as well as in solution. On a surface, the association coefficient is about twice that in solution. The difference is probably not significant. Finally, the fully octameric form of the protein that does not contain a hexa-his N-terminal peptide does not bind to a silicon oxynitride surface, does not bind to an Au-nanoparticle surface, does not bind to a surface coated with Ni-NTA nor does it bind to a surface coated with DPgn. The likelihood is great that the enolase species on the surface of Streptococcus pyogenes is an x-mer of the native octamer.

Oligomeric forms of bacterial malate dehydrogenase: a study of the enzyme from the phototrophic non-sulfur bacterium Rhodovulum steppense A-20s.

Malate dehydrogenase (EC 1.1.1.37) was purified to homogeneity from the phototrophic purple non-sulfur bacterium Rhodovulum steppense A-20s. According to gel-chromatography and electrophoretic studies, malate dehydrogenase is present as a dimer, tetramer and octamer depending on cultivation conditions. In phototrophic aerobic conditions only the tetrameric form was present, in chemotrophic aerobic conditions all three forms were detected, while in the absence of oxygen the octameric form disappeared. The malate dehydrogenase oligomers are encoded by a single gene and composed of the same 35kDa polypeptide but differ in pH and temperature optimum, in affinities to malate, oxaloacetate, NADH and NAD+ and in regulation by cations and citrate. By modulating the cultivation conditions, it has been established that the dimer participates in the glyoxylate cycle; the tetramer operates in the tricarboxylic acid cycle, and the octamer may be involved in the adaptation to oxidative stress.

The C-Domain of Oleuropein β-Glucosidase Assists in Protein Folding and Sequesters the Enzyme in Nucleus.

Oleuropein, a terpene-derived glycosylated secoiridoid biosynthesized exclusively by members of the Oleaceae family, is involved in a two-component defense system comprising a β-glucosidase that activates oleuropein into a toxic glutaraldehyde-like structure. Oleuropein and its deglycosylated derivatives have high pharmaceutical interest. In this study we determined that the in planta heterologous expressed OeGLU, an oleuropein-specific β-glucosidase from olive (Olea europaea), had enzymatic kinetics similar to the olive native enzyme. The C terminus encompassing the nuclear localization signal sequesters the enzyme in the nucleus, and predetermines the protein-protein recognition and homodimerization. Biochemical analysis revealed that OeGLU is a homomultimer with high Mr In silico prediction modeling of the complex structure and bimolecular fluorescence complementation analyses revealed that the C terminus of OeGLU is essential for the proper assembly of an octameric form, a key conformational feature that determines the activity of the enzyme. Our results demonstrate that intrinsic characteristics of the OeGLU ensure separation from oleuropein and keep the dual-partner defensive system conditionally inactive. Upon cell destruction, the dual-partner defense system is activated and olive massively releases the arsenal of defense.

Disrupting a key hydrophobic pair in the oligomerization interface of the actinoporins impairs their pore-forming activity.

Crystallographic data of the dimeric and octameric forms of fragaceatoxin C (FraC) suggested the key role of a small hydrophobic protein-protein interaction surface for actinoporins oligomerization and pore formation in membranes. However, site-directed mutagenesis studies supporting this hypothesis for others actinoporins are still lacking. Here, we demonstrate that disrupting the key hydrophobic interaction between V60 and F163 (FraC numbering scheme) in the oligomerization interface of FraC, equinatoxin II (EqtII), and sticholysin II (StII) impairs the pore formation activity of these proteins. Our results allow for the extension of the importance of FraC protein-protein interactions in the stabilization of the oligomeric intermediates of StII and EqtII pointing out that all of these proteins follow a similar pathway of membrane disruption. These findings support the hybrid pore proposal as the universal model of actinoporins pore formation. Moreover, we reinforce the relevance of dimer formation, which appears to be a functional intermediate in the assembly pathway of some different pore-forming proteins.

Cloning, expression and characterization of the gene encoding the enolase from Fusobacterium nucleatum

The gene encoding enolase from Fusobacterium nucleatum (FnENO) was cloned and analyzed for the first time. The gene comprises of 1302 nucleotide base pairs and encodes 433 amino acids. The gene sequence alignment demonstrated the presence of several distinct insertions and deletions, compared with the human enzyme. The gene for recombinant FnENO was inserted into the pLATE 31 vector system and expressed in E. coli BL21(DE3) cells as a soluble protein. The protein was purified by affinity chromatography using a Ni-NTA agarose matrix and shown on SDS-PAGE to be a 46 kDa protein. The molecular weight of the octameric form of the purified recombinant protein was determined as being 375 kDa by size exclusion chromatography. Optimal enzyme activity was observed at pH 8.5 and the enzyme remained stable at a range of different temperatures from 30 to 60°C. Using 2-phosphoglyceric acid as substrate for the purified enzyme, KM, kcat and kcat/KM were determined as 0.48 mM, 20.4 s–1 and 4.22 × 104 M–1s–1, respectively. Potential drug binding sites of FnENO were detected using homology modeling. These data could facilitate the design of new inhibitors of F. nucleatum which has already been shown to be resistant to several known antibiotics.

Octameric structure of Staphylococcus aureus enolase in complex with phosphoenolpyruvate.

Staphylococcus aureus is a Gram-positive bacterium with strong pathogenicity that causes a wide range of infections and diseases. Enolase is an evolutionarily conserved enzyme that plays a key role in energy production through glycolysis. Additionally, enolase is located on the surface of S. aureus and is involved in processes leading to infection. Here, crystal structures of Sa_enolase with and without bound phosphoenolpyruvate (PEP) are presented at 1.6 and 2.45 Å resolution, respectively. The structure reveals an octameric arrangement; however, both dimeric and octameric conformations were observed in solution. Furthermore, enzyme-activity assays show that only the octameric variant is catalytically active. Biochemical and structural studies indicate that the octameric form of Sa_enolase is enzymatically active in vitro and likely also in vivo, while the dimeric form is catalytically inactive and may be involved in other biological processes.

The Protein Acetyltransferase PatZ from Escherichia coli Is Regulated by Autoacetylation-induced Oligomerization

Lysine acetylation is an important post-translational modification in the metabolic regulation of both prokaryotes and eukaryotes. In Escherichia coli, PatZ (formerly YfiQ) is the only known acetyltransferase protein and is responsible for acetyl-CoA synthetase acetylation. In this study, we demonstrated PatZ-positive cooperativity in response to acetyl-CoA and the regulation of acetyl-CoA synthetase activity by the acetylation level. Furthermore, functional analysis of an E809A mutant showed that the conserved glutamate residue is not relevant for the PatZ catalytic mechanism. Biophysical studies demonstrated that PatZ is a stable tetramer in solution and is transformed to its octameric form by autoacetylation. Moreover, this modification is reversed by the sirtuin CobB. Finally, an in silico PatZ tetramerization model based on hydrophobic and electrostatic interactions is proposed and validated by three-dimensional hydrodynamic analysis. These data reveal, for the first time, the structural regulation of an acetyltransferase by autoacetylation in a prokaryotic organism.

Potent suppression of c-di-GMP synthesis via I-site allosteric inhibition of diguanylate cyclases with 2′-F-c-di-GMP

Cyclic-di-GMP (c-di-GMP) is a central regulator of bacterial behavior. Various studies have implicated c-di-GMP in biofilm formation and virulence factor production in multitudes of bacteria. Hence it is expected that the disruption of c-di-GMP signaling could provide an effective means to disrupt biofilm and/or virulence factor formation in several bacteria of clinical relevance. C-di-GMP achieves the regulation of bacterial phenotype via binding to several effector molecules including transcription factors, enzymes and riboswitches. Crystal structure analyses of c-di-GMP effector molecules, in complex with the ligand, reveal that various classes of c-di-GMP receptors recognize this dinucleotide using different sets of recognition elements. Therefore, it is plausible that different analogues of c-di-GMP could be used to selectively modulate a specific class of c-di-GMP binding receptors, and hence modulate the bacterial phenotype. Thus far only a detailed study of the differential binding of c-di-GMP analogues to riboswitches, but not proteins, has been reported. In this report, we prepared various 2′-modified analogues of c-di-GMP and studied both polymorphisms of these analogues using DOSY NMR and the binding to several effector proteins, such as PilZ-containing proteins, diguanylate cyclases (DGC) containing I-sites, and phoshphodiesterases (PDE). 2′-Modification of c-di-GMP did not adversely affect the propensity to form higher aggregates, such as octameric forms, in the presence of potassium salts. Interestingly, we find that the selective binding to different classes of c-di-GMP binding proteins could be achieved with the 2′-modified analogues and that 2′-F analogue of c-di-GMP binds to the I-site of DGCs better (four times) than the native dinucleotide, c-di-GMP, whereas c-di-GMP binds to PDEs better (10 times) than 2′-F-c-di-GMP. 2′-F-c-di-GMP potently inhibits c-di-GMP synthesis by DGCs and hence raises the potential that cell permeable analogues of 2′-F-c-di-GMP could be used to disrupt c-di-GMP signaling in bacteria.

Structure of a filament of stacked octamers of human DMC1 recombinase.

Eukaryal DMC1 proteins play a central role in homologous recombination in meiosis by assembling at the sites of programmed DNA double-strand breaks and carrying out a search for allelic DNA sequences located on homologous chromatids. They are close homologs of eukaryal Rad51 and archaeal RadA proteins and are remote homologs of bacterial RecA proteins. These recombinases (also called DNA strand-exchange proteins) promote a pivotal strand-exchange reaction between homologous single-stranded and double-stranded DNA substrates. An octameric form of a truncated human DMC1 devoid of its small N-terminal domain (residues 1-83) has been crystallized. The structure of the truncated DMC1 octamer is similar to that of the previously reported full-length DMC1 octamer, which has disordered N-terminal domains. In each protomer, only the ATP cap regions (Asp317-Glu323) show a noticeable conformational difference. The truncated DMC1 octamers further stack with alternate polarity into a filament. Similar filamentous assemblies of DMC1 have been observed to form on DNA by electron microscopy.

γ-Hemolysin oligomeric structure and effect of its formation on supported lipid bilayers: An AFM Investigation

γ-Hemolysins are bicomponent β-barrel pore forming toxins produced by Staphylococcus aureus as water-soluble monomers, which assemble into oligomeric pores on the surface of lipid bilayers. Here, after investigating the oligomeric structure of γ-hemolysins on supported lipid bilayers (SLBs) by atomic force microscopy (AFM), we studied the effect produced by this toxin on the structure of SLBs. We found that oligomeric structures with different number of monomers can assemble on the lipid bilayer being the octameric form the stablest one. Moreover, in this membrane model we found that γ-hemolysins can form clusters of oligomers inducing a curvature in the lipid bilayer, which could probably enhance the aggressiveness of these toxins at high concentrations.

Characterization of a novel Agrobacterium tumefaciens Galactarolactone Cycloisomerase Enzyme for Direct Conversion of d-Galactarolactone to 3-Deoxy-2-keto-l-threo-hexarate

Microorganisms use different pathways for D-galacturonate catabolism. In the known microbial oxidative pathway, D-galacturonate is oxidized to D-galactarolactone, the lactone hydrolyzed to galactarate, which is further converted to 3-deoxy-2-keto-hexarate and α-ketoglutarate. We have shown recently that Agrobacterium tumefaciens strain C58 contains an uronate dehydrogenase (At Udh) that oxidizes D-galacturonic acid to D-galactarolactone. Here we report identification of a novel enzyme from the same A. tumefaciens strain, which we named Galactarolactone cycloisomerase (At Gci) (E.C. 5.5.1.-), for the direct conversion of the D-galactarolactone to 3-deoxy-2-keto-hexarate. The At Gci enzyme is 378 amino acids long and belongs to the mandelate racemase subgroup in the enolase superfamily. At Gci was heterologously expressed in Escherichia coli, and the purified enzyme was found to exist as an octameric form. It is active both on D-galactarolactone and D-glucarolactone, but does not work on the corresponding linear hexaric acid forms. The details of the reaction mechanism were further studied by NMR and optical rotation demonstrating that the reaction product of At Gci from D-galactaro-1,4-lactone and D-glucaro-1,4-lactone conversion is in both cases the L-threo form of 3-deoxy-2-keto-hexarate.

Construction and Analyses of Tetrameric Forms of Yeast NAD+-Specific Isocitrate Dehydrogenase

Yeast NAD(+)-specific isocitrate dehydrogenase (IDH) is an octameric enzyme composed of four heterodimers of regulatory IDH1 and catalytic IDH2 subunits. The crystal structure suggested that the interactions between tetramers in the octamer are restricted to defined regions in IDH1 subunits from each tetramer. Using truncation and mutagenesis, we constructed three tetrameric forms of IDH. Truncation of five residues from the amino terminus of IDH1 did not alter the octameric form of the enzyme, but this truncation with an IDH1 G15D or IDH1 D168K residue substitution produced tetrameric enzymes as assessed by sedimentation velocity ultracentrifugation. The IDH1 G15D substitution in the absence of any truncation of IDH1 was subsequently found to be sufficient for production of a tetrameric enzyme. The tetrameric forms of IDH exhibited ∼50% reductions in V(max) and in cooperativity with respect to isocitrate relative to those of the wild-type enzyme, but they retained the property of allosteric activation by AMP. The truncated (-5)IDH1/IDH2 and tetrameric enzymes were much more sensitive than the wild-type enzyme to inhibition by the oxidant diamide and concomitant formation of a disulfide bond between IDH2 Cys-150 residues. Binding of ligands reduced the sensitivity of the wild-type enzyme to diamide but had no effect on inhibition of the truncated or tetrameric enzymes. These results suggest that the octameric structure of IDH has in part evolved for regulation of disulfide bond formation and activity by ensuring the proximity of the amino terminus of an IDH1 subunit of one tetramer to the IDH2 Cys-150 residues in the other tetramer.

Engineered Rings of Mixed Yeast Lsm Proteins Show Differential Interactions with Translation Factors and U-Rich RNA

The Lsm proteins organize as heteroheptameric ring assemblies capable of binding RNA substrates and ancillary protein factors. We have constructed simplified Lsm polyproteins that organize as multimeric ring structures as analogues of the functional Lsm complexes. Polyproteins Lsm[2+3], Lsm[4+1], and Lsm[5+6] incorporate natural sequence extensions as linker peptides between the core Lsm domains. In solution, the recombinant products organize as stable ring oligomers (75 A wide, 20 A pores) in discrete tetrameric and octameric forms. Following immobilization, the polyproteins successfully act as affinity pull-down ligands for proteins within yeast lysate, including native Lsm proteins. Interaction partners were consistent with current models of the mixed Lsm ring assembly in vivo but also suggest that dynamic rearrangements of Lsm protein complexes can occur. The Lsm polyprotein ring complexes were seen in gel shift assays to have a preference for U-rich RNA sequences, with tightest binding measured for Lsm[2+3] with U(10). Polyprotein rings containing truncated forms of Lsm1 and Lsm4 were found to associate with translation, initiation, and elongation protein factors in an RNA-dependent manner. Our findings suggest Lsm1 and/or Lsm4 can interact with translationally active mRNA.

Coordination of genomic structure and transcription by the main bacterial nucleoid‐associated protein HU

The histone-like protein HU is a highly abundant DNA architectural protein that is involved in compacting the DNA of the bacterial nucleoid and in regulating the main DNA transactions, including gene transcription. However, the coordination of the genomic structure and function by HU is poorly understood. Here, we address this question by comparing transcript patterns and spatial distributions of RNA polymerase in Escherichia coli wild-type and hupA/B mutant cells. We demonstrate that, in mutant cells, upregulated genes are preferentially clustered in a large chromosomal domain comprising the ribosomal RNA operons organized on both sides of OriC. Furthermore, we show that, in parallel to this transcription asymmetry, mutant cells are also impaired in forming the transcription foci-spatially confined aggregations of RNA polymerase molecules transcribing strong ribosomal RNA operons. Our data thus implicate HU in coordinating the global genomic structure and function by regulating the spatial distribution of RNA polymerase in the nucleoid.