Oligomeric states of bacteriophage T7 gene 4 primase/helicase

Electron microscopic and crystallographic data have shown that the gene 4 primase/helicase encoded by bacteriophage T7 can form both hexamers and heptamers. After cross-linking with glutaraldehyde to stabilize the oligomeric protein, hexamers and heptamers can be distinguished either by negative stain electron microscopy or electrophoretic analysis using polyacrylamide gels. We find that hexamers predominate in the presence of either dTTP or beta,gamma-methylene dTTP whereas the ratio between hexamers and heptamers is nearly the converse in the presence of dTDP. When formed, heptamers are unable to efficiently bind either single-stranded DNA or double-stranded DNA. We postulate that a switch between heptamer to hexamer may provide a ring-opening mechanism for the single-stranded DNA binding pathway. Accordingly, we observe that in the presence of both nucleoside di- and triphosphates the gene 4 protein exists as a hexamer when bound to single-stranded DNA and as a mixture of heptamer and hexamer when not bound to single-stranded DNA. Furthermore, altering regions of the gene 4 protein postulated to be conformational switches for dTTP-dependent helicase activity leads to modulation of the heptamer to hexamer ratio.     

Crystal structures of Lsm3, Lsm4 and Lsm5/6/7 from Schizosaccharomyces pombe

Sm-like (Lsm) proteins are ubiquitous and function in many aspects of RNA metabolism, including pre-mRNA splicing, nuclear RNA processing, mRNA decay and miRNA biogenesis. Here three crystal structures including Lsm3, Lsm4 and Lsm5/6/7 sub-complex from S. pombe are reported. These structures show that all the five individual Lsm subunits share a conserved Sm fold, and Lsm3, Lsm4, and Lsm5/6/7 form a heptamer, a trimer and a hexamer within the crystal lattice, respectively. Analytical ultracentrifugation indicates that Lsm3 and Lsm5/6/7 sub-complex exist in solution as a heptamer and a hexamer, respectively while Lsm4 undergoes a dynamic equilibrium between monomer and trimer in solution. RNA binding assays show that Lsm2/3 and Lsm5/6/7 bind to oligo(U) whereas no RNA binding is observed for Lsm3 and Lsm4. Analysis of the inter-subunit interactions in Lsm5/6/7 reveals the organization order among Lsm5, Lsm6 and Lsm7.     

Hemocyanins in spiders. XXII. Range of allosteric interaction in a four-hexamer hemocyanin. Co-operativity and Bohr effect in dissociation intermediates

The range of allosteric interaction in the 24-meric hemocyanin from the tarantula Eurypelma californicum was studied by measuring the oxygen-binding properties of defined oligomeric fragments. Dissociation intermediates comprising 19, 12, 7 or 6 subunits were obtained by incubation of native hemocyanin with 10 mM-cysteine at pH 4.4, with 40 mM-dithiothreitol at pH 7 or 8, by short-term alkaline (pH 9.6) treatment or by treatment with 4 M-urea. These could be stabilized by returning to neutral buffer conditions and, in the case of the dodecamer, by carboxymethylation. Conditions were chosen so that part of the starting material remained intact to serve as control in the oxygen-binding measurements. Oxygen equilibrium curves were obtained by a very sensitive fluorimetric/polarographic method. Oxygen affinity and the magnitude of the Bohr effect remain constant from the hexamer up to the control four-hexamer. Co-operativity, in contrast, increases with aggregate size in two steps: n (hexamer) = n (heptamer) less than n (dodecamer) = n (19-mer) less than n (4-hexamer). The characteristic pH-dependence of nH is absent in the hexa- and heptamer but is weakly indicated in the dodecamer, and fully established in the four-hexamer. Results from different preparations are highly consistent, if nH is expressed as a percentage of the control values. Full co-operativity is reached only in the four-hexamer, disproving the dodecameric half-molecule (the smallest repeating unit) as the allosteric unit. The stepwise increase in co-operativity appears to be correlated with higher levels of symmetry in the hierarchy of quaternary structure.     

Visualization of Sparsely-populated Lower-order Oligomeric States of Human Mitochondrial Hsp60 by Cryo-electron Microscopy

Human mitochondrial Hsp60 (mtHsp60) is a class I chaperonin, 51% identical in sequence to the prototypical E. coli chaperonin GroEL. mtHsp60 maintains the proteome within the mitochondrion and is associated with various neurodegenerative diseases and cancers. The oligomeric assembly of mtHsp60 into heptameric ring structures that enclose a folding chamber only occurs upon addition of ATP and is significantly more labile than that of GroEL, where the only oligomeric species is a tetradecamer. The lability of the mtHsp60 heptamer provides an opportunity to detect and visualize lower-order oligomeric states that may represent intermediates along the assembly/disassembly pathway. Using cryo-electron microscopy we show that, in addition to the fully-formed heptamer and an “inverted” tetradecamer in which the two heptamers associate via their apical domains, thereby blocking protein substrate access, well-defined lower-order oligomeric species, populated at less than 6% of the total particles, are observed. Specifically, we observe open trimers, tetramers, pentamers and hexamers (comprising ∼4% of the total particles) with rigid body rotations from one subunit to the next within ∼1.5–3.5° of that for the heptamer, indicating that these may lie directly on the assembly/disassembly pathway. We also observe a closed-ring hexamer (∼2% of the particles) which may represent an off-pathway species in the assembly/disassembly process in so far that conversion to the mature heptamer would require the closed-ring hexamer to open to accept an additional subunit. Lastly, we observe several classes of tetramers where additional subunits characterized by fuzzy electron density are caught in the act of oligomer extension.     

The effect of homologos amini acid replacement on the conformation of oligopeptides. III. CD studies on co-oligopeptides of methionine and valine or methionine and glycine in organic solution

A conformational analyis of co-ologopeptides containing methionine and valine or methionine and glycine was carried out using circular dichrosim. The oligopeptides containing valine and methionine (dimer to hexamer) are disorder in hexafluoropropane diol·0.5 H₂O and trimethyl phospate but become helical in trifluoroethanol at the heptamer. The CD spectra for hesamers and heptamers containing methionine or methionine and one valine give evidience that one valyl residue can be inserted into these peptides wothout affecting their secondry structure. Co-oligomethionines. The effect of a glycyl residue are generally less ordered than the analogous homo-oligomethionines. The effect of a glycl residue on the structure of the longer oligopeptides depends on its position in the chain. When inserted in the center of a hexamer or heptamer, the single glycyl residue destabilizes the ordered secondry structures in solution. Finally, evidence is presented that the CD patterns observed for various pentamers and hexamers are consistent with some order at these chain lenghts.     

The active site of yeast endo-polygalacturonase contains seven subsites

The rate of hydrolysis of oligomers by the endopolygalacturonase of yeast is in the order: heptamer > hexamer > pentamer > tetramer. This suggests that the active site accommodates at least 7 units. Since the heptamer disappears concurrently with the bulk of larger oligomers, the maximum number of units appears to be 7. The release of labelled (unsaturated, or ³H labelled and reduced) end units from larger substrate is interpreted to indicate that the enzyme interacts with 3 saccharide units toward the reducing end from the bond to be broken, and with 4 units toward the non-reducing end. The relative affinities for the enzyme of saccharide units in various positions are unequal, as indicated by the very low relative rate of monomer production from the hydrolysis of hexamer and pentamer, and the apparently unequal probability of two other modes of hexamer hydrolysis [(tetramer + dimer) = 2.5 (trimer + trimer)].     

Hexamer to monomer equilibrium of E. coli Hfq in solution and its impact on RNA annealing

The bacterial Sm-like protein Hfq forms a ring-shaped homo-hexamer that is necessary for Hfq to bind nucleic acids and to act in small noncoding RNA regulation. Using semi-native gels and fluorescence anisotropy, we show that Hfq undergoes a cooperative conformational change from monomer to hexamer around 1 μM protein, which is comparable to the in vivo concentration of Hfq and above the dissociation constant of the Hfq hexamer from many RNA substrates. Above 2 μM protein, Hfq hexamers associate in high-molecular-weight complexes. Mutations that impair RNA binding to the proximal face strongly destabilize the hexamer, while the mutation R16A near the outer rim prevents hexamer association. Stopped-flow fluorescence resonance energy transfer experiments showed that Hfq subunits interact within a few seconds, suggesting that Hfq monomers, hexamers and multi-hexamer complexes are in dynamic equilibrium. Finally, we show that Hfq is most active in RNA annealing when the hexamer is present. These results suggest that RNA binding is coupled to hexamer assembly and that the biochemical activity of Hfq reflects the equilibrium between different quaternary structures.     

Critical chain length for helix formation in L-methionine oligopeptides

The circular dichroism of a series of L -methionine oligopeptides [BOC-(Met)_n-OMe] was examined in trifluoroethanol and hexafluoroacetone sesquihydrate. The results indicate that the trimer through the hexamer exists predominantly in disordered conformations in these solvents. An abrupt change in the CD pattern at the heptamer in trifluoroethanol suggests that L -methionine oligopeptides begin forming helices at this chain length.     

3' UTR seed matches, but not overall identity, are associated with RNAi off-targets

Off-target gene silencing can present a notable challenge in the interpretation of data from large-scale RNA interference (RNAi) screens. We performed a detailed analysis of off-targeted genes identified by expression profiling of human cells transfected with small interfering RNA (siRNA). Contrary to common assumption, analysis of the subsequent off-target gene database showed that overall identity makes little or no contribution to determining whether the expression of a particular gene will be affected by a given siRNA, except for near-perfect matches. Instead, off-targeting is associated with the presence of one or more perfect 3' untranslated region (UTR) matches with the hexamer or heptamer seed region (positions 2-7 or 2-8) of the antisense strand of the siRNA. These findings have strong implications for future siRNA design and the application of RNAi in high-throughput screening and therapeutic development.     

Cryo-EM Analysis Reveals Structural Basis of Helicobacter pylori VacA Toxin Oligomerization

Helicobacter pylori colonizes the human stomach and contributes to the development of gastric cancer and peptic ulcer disease. H. pylori secretes a pore-forming toxin called vacuolating cytotoxin A (VacA), which contains two domains (p33 and p55) and assembles into oligomeric structures. Using single-particle cryo-electron microscopy, we have determined low-resolution structures of a VacA dodecamer and heptamer, as well as a 3.8-Å structure of the VacA hexamer. These analyses show that VacA p88 consists predominantly of a right-handed beta-helix that extends from the p55 domain into the p33 domain. We map the regions of p33 and p55 involved in hexamer assembly, model how interactions between protomers support heptamer formation, and identify surfaces of VacA that likely contact membrane. This work provides structural insights into the process of VacA oligomerization and identifies regions of VacA protomers that are predicted to contact the host cell surface during channel formation.     

Phycocyanin aggregation. A small angle neutron scattering and size exclusion chromatographic study

The influence of environmental factors on the aggregation properties of phycocyanin from Synechocystis 6701 was studied by small angle neutron scattering and high-pressure size-exclusion liquid chromatography. Phycocyanin was found to exist in a reversible equilibrium between the monomer, trimer and hexamer forms. The distribution of the protein between these oligomers is determined by the pH, buffer composition and ionic strength of the medium, and protein concentration. Phycocyanin was in a stable hexameric state at pH 5.0 to 6.0 at a concentration of 1 to 10 mg/ml, and was primarily in a trimeric state at pH 8.0 at a concentration of about 5 mg/ml. Comparison of the small angle scattering data with the computed scattering curve for a hollow cylinder was used to determine the dimensions of the best-fit model by a least-squares fitting procedure. The outer radius, inner radius and height of the phycocyanin hexamer were found to be 54.1, 12.0 and 61.4 A (1 A = 0.1 nm), respectively, and the corresponding dimensions for the trimer were 54.5, 14.0 and 33.0 A. The molecular weight ratio for phycocyanin hexamer was determined to be 217,000. The dimensions and molecular weight ratios of phycocyanin from Synechocystis 6701 obtained by solution scattering are similar to the values for Mastigocladus laminosus obtained by X-ray crystallography.     

Structural studies of the archaeal MCM complex in different functional states

The primary candidate for the eukaryotic replicative helicase is the MCM2-7 complex, a hetero-oligomer formed by six AAA+ paralogous polypeptides. A simplified model for structure-function studies is the homo-oligomeric orthologue from the archaeon Methanothermobacter thermoautotrophicus. The crystal structure of the DNA-interacting N-terminal domain of this homo-oligomer revealed a double hexamer in a head-to-head configuration; single-particle electron microscopy studies have shown that the full-length protein complex can form both single and double rings, in which each ring can consist of a cyclical arrangement of six or seven subunits. Using single-particle techniques and especially multivariate statistical symmetry analysis, we have assessed the changes in stoichiometry that the complex undergoes when treated with various nucleotide analogues or when binding a double-stranded DNA fragment. We found that the binding of nucleotides or of double-stranded DNA leads to the preferred formation of double-ring structures. Specifically, the protein complex is present as a double heptamer when treated with a nucleotide analogue, but it is rather found as a double hexamer when complexed with double-stranded DNA. The possible physiological role of the various stoichiometries of the complex is discussed in the light of the proposed mechanisms of helicase activity.     

Degradation of a Sodium Acrylate Oligomer by an Arthrobacter sp

Arthrobacter sp. strain NO-18 was first isolated from soil as a bacterium which could degrade the sodium acrylate oligomer and utilize it as the sole source of carbon. When 0.2% (wt/wt) oligomer was added to the culture medium, the acrylate oligomer was found to be degraded by 70 to 80% in 2 weeks, using gel permeation chromatography. To determine the maximum molecular weight for biodegradation, the degradation test was done with the hexamer, heptamer, and octamer, which were separated from the oligomer mixture by fractional gel permeation chromatography. The hexamer and heptamer were consumed to the extents of 58 and 36%, respectively, in 2 weeks, but the octamer was not degraded. Oligomers with three different terminal groups were synthesized to examine the effect of the different terminal groups on biodegradation, but few differences were found. Arthrobacter sp. NO-18 assimilated acrylic acid, propionic acid, glutaric acid, 2-methylglutaric acid, and 1,3,5-pentanetricarboxylic acid. Degradation of the acrylic unit structure by this strain is discussed.     

Two different oligomeric states of the RuvB branch migration motor protein as revealed by electron microscopy

In prokaryotes, the RuvA, B, and C proteins play major roles at the late stage of DNA homologous recombination, where RuvB complexed with RuvA acts as an ATP-dependent motor for branch migration. The oligomeric structures of negatively stained and frozen hydrated RuvB from Thermus thermophilus HB8 were investigated by electron microscopy. RuvB oligomers free of DNA formed a ring structure of about 14 nm in diameter. The averaged top view image clearly indicated a sevenfold symmetry, suggesting that it exists as a heptamer. The RuvB oligomers complexed with duplex DNA formed a smaller ring of about 13 nm in diameter. The averaged top view images represented a sixfold symmetry. This difference in oligomerization indicates that the oligomeric structure of RuvB may convert from a heptamer to a hexamer upon DNA binding. In addition, this finding provides the lesson that great care should be taken in investigating the subunit organizations of DNA binding proteins, because their oligomeric states are more sensitive to DNA interactions than expected.     

RNA heptamers that direct RNA cleavage by mammalian tRNA 3' processing endoribonuclease.

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) can recognize and cleave any target RNA that forms a precursor tRNA-like complex with another RNA. Various sets of RNA molecules were tested to identify the smallest RNA that can direct target RNA cleavage by 3' tRNase. A 3' half tRNAArgwas cleaved efficiently by 3' tRNase in the presence of small 5' half tRNAArgvariants, the D stem-loop region of which was partially deleted. Remarkably, 3' tRNase also cleaved the 3' half tRNAArgin the presence of a 7 nt 5' tRNAArg composed only of the acceptor stem region with a catalytic efficiency comparable with that of cleavage directed by an intact 5' half tRNAArg. The catalytic efficiency of cleavage directed by the heptamer decreased as the stability of the T stem-loop structures of 3' half tRNAArg variants decreased. No heptamer-directed cleavage of a 3' half tRNAArg without T stem base pairs was detected. A heptamer also directed cleavage of an HIV-1 RNA containing a stable hairpin structure. These findings suggest that in the presence of an RNA heptamer, 3' tRNase can discriminate and eliminate target RNAs that possess a stable hairpin adjacent to the heptamer binding sequence from a large complex RNA pool.