Crystal structure of the Holliday junction DNA in complex with a single RuvA tetramer

In the major pathway of homologous DNA recombination in prokaryotic cells, the Holliday junction intermediate is processed through its association with RuvA, RuvB, and RuvC proteins. Specific binding of the RuvA tetramer to the Holliday junction is required for the RuvB motor protein to be loaded onto the junction DNA, and the RuvAB complex drives the ATP-dependent branch migration. We solved the crystal structure of the Holliday junction bound to a single Escherichia coli RuvA tetramer at 3.1-Å resolution. In this complex, one side of DNA is accessible for cleavage by RuvC resolvase at the junction center. The refined junction DNA structure revealed an open concave architecture with a four-fold symmetry. Each arm, with B-form DNA, in the Holliday junction is predominantly recognized in the minor groove through hydrogen bonds with two repeated helix-hairpin-helix motifs of each RuvA subunit. The local conformation near the crossover point, where two base pairs are disrupted, suggests a possible scheme for successive base pair rearrangements, which may account for smooth Holliday junction movement without segmental unwinding.

Transcription coupled repair of 8-oxoguanine in murine cells: The Ogg1 protein is required for repair in nontranscribed sequences but not in transcribed sequences

To assess the role of the Ogg1 DNA glycosylase in the transcription-coupled repair (TCR) of the mutagenic lesion, 7,8-dihydro-8oxoguanine (8-OxoG), we have investigated the removal of this lesion in wild-type and ogg1−/− null mouse embryo fibroblast (MEF) cell lines. We used nonreplicating plasmids containing a single 8-OxoG·C base pair in a different assay that allowed us to study the removal of 8-OxoG located in a transcribed sequence (TS) or in a nontranscribed sequence (NTS). The results show that the removal of 8-OxoG in a wild-type MEF cell line is faster in the TS than in the NTS, indicating TCR of 8-OxoG in murine cells. In the homozygous ogg1−/− MEF cell line, 8-OxoG was not removed from the NTS whereas there was still efficient 8-OxoG repair in the TS. Expression of the mouse Ogg1 protein in the homozygous ogg1−/− cell line restored the ability to remove 8-OxoG in the NTS. Therefore, we have demonstrated that Ogg1 is essential for the repair of 8-OxoG in the NTS but is not required in the TS. These results indicate the existence of an Ogg1-independent pathway for the TCR of 8-OxoG in vivo.

Delivery of a stringent dimerizer-regulated gene expression system in a single retroviral vector

Small molecule-regulated transcription has broad utility and would benefit from an easily delivered self-contained regulatory cassette capable of robust, tightly controlled target gene expression. We describe the delivery of a modified dimerizer-regulated gene expression system to cells on a single retrovirus. A transcription factor cassette responsive to the natural product dimerizer rapamycin was optimized for retroviral delivery by fusing a highly potent chimeric activation domain to the rapamycin-binding domain of FKBP-rapamycin-associated protein (FRAP). This improvement led to an increase in both the potency and maximal levels of gene expression induced by rapamycin, or nonimmunosuppressive rapamycin analogs. The modified transcription factor cassette was incorporated along with a target gene into a single rapamycin-responsive retrovirus. Cell pools stably transduced with the single virus system displayed negligible basal expression and gave induction ratios of at least three orders of magnitude in the presence of rapamycin or a nonimmunosuppressive rapamycin analog. Levels of induced gene expression were comparable to those obtained with the constitutive retroviral long terminal repeat and the single virus system performed well in four different mammalian cell lines. Regulation with the dimerizer-responsive retrovirus was tight enough to allow the generation of cell lines displaying inducible expression of the highly toxic diphtheria toxin A chain gene. The ability to deliver the tightly inducible rapamycin system in a single retrovirus should facilitate its use in the study of gene function in a broad range of cell types.

Free energy of burying hydrophobic residues in the interface between protein subunits

We have obtained an experimental estimate of the free energy change associated with variations at the interface between protein subunits, a subject that has raised considerable interest since the concept of accessible surface area was introduced by Lee and Richards [Lee, B. & Richards, F. M. (1971) J. Mol. Biol. 55, 379–400]. We determined by analytical ultracentrifugation the dimer–tetramer equilibrium constant of five single and three double mutants of human Hb. One mutation is at the stationary α1β1 interface, and all of the others are at the sliding α1β2 interface where cleavage of the tetramer into dimers and ligand-linked allosteric changes are known to occur. A surprisingly good linear correlation between the change in the free energy of association of the mutants and the change in buried hydrophobic surface area was obtained, after corrections for the energetic cost of losing steric complementarity at the αβ dimer interface. The slope yields an interface stabilization free energy of −15 ± 1.2 cal/mol upon burial of 1 Å2 of hydrophobic surface, in very good agreement with the theoretical estimate given by Eisenberg and McLachlan [Eisenberg, D. & McLachlan, A. D. (1986) Nature (London) 319, 199–203].

A single receptor encoded by vzg-1/lpA1/edg-2 couples to G proteins and mediates multiple cellular responses to lysophosphatidic acid

Extracellular lysophosphatidic acid (LPA) produces diverse cellular responses in many cell types. Recent reports of several molecularly distinct G protein-coupled receptors have raised the possibility that the responses to LPA stimulation could be mediated by the combination of several uni-functional receptors. To address this issue, we analyzed one receptor encoded by ventricular zone gene-1 (vzg-1) (also referred to as lpA1/edg-2) by using heterologous expression in a neuronal and nonneuronal cell line. VZG-1 expression was necessary and sufficient in mediating multiple effects of LPA: [3H]-LPA binding, G protein activation, stress fiber formation, neurite retraction, serum response element activation, and increased DNA synthesis. These results demonstrate that a single receptor, encoded by vzg-1, can activate multiple LPA-dependent responses in cells from distinct tissue lineages.

Solvent dependence of dynamic transitions in protein solutions

A transition as a function of increasing temperature from harmonic to anharmonic dynamics has been observed in globular proteins by using spectroscopic, scattering, and computer simulation techniques. We present here results of a dynamic neutron scattering analysis of the solvent dependence of the picosecond-time scale dynamic transition behavior of solutions of a simple single-subunit enzyme, xylanase. The protein is examined in powder form, in D2O, and in four two-component perdeuterated single-phase cryosolvents in which it is active and stable. The scattering profiles of the mixed solvent systems in the absence of protein are also determined. The general features of the dynamic transition behavior of the protein solutions follow those of the solvents. The dynamic transition in all of the mixed cryosolvent–protein systems is much more gradual than in pure D2O, consistent with a distribution of energy barriers. The differences between the dynamic behaviors of the various cryosolvent protein solutions themselves are remarkably small. The results are consistent with a picture in which the picosecond-time scale atomic dynamics respond strongly to melting of pure water solvent but are relatively invariant in cryosolvents of differing compositions and melting points.

Unbinding forces of single antibody-antigen complexes correlate with their thermal dissociation rates

Point mutants of three unrelated antifluorescein antibodies were constructed to obtain nine different single-chain Fv fragments, whose on-rates, off-rates, and equilibrium binding affinities were determined in solution. Additionally, activation energies for unbinding were estimated from the temperature dependence of the off-rate in solution. Loading rate-dependent unbinding forces were determined for single molecules by atomic force microscopy, which extrapolated at zero force to a value close to the off-rate measured in solution, without any indication for multiple transition states. The measured unbinding forces of all nine mutants correlated well with the off-rate in solution, but not with the temperature dependence of the reaction, indicating that the same transition state must be crossed in spontaneous and forced unbinding and that the unbinding path under load cannot be too different from the one at zero force. The distance of the transition state from the ground state along the unbinding pathway is directly proportional to the barrier height, regardless of the details of the binding site, which most likely reflects the elasticity of the protein in the unbinding process. Atomic force microscopy thus can be a valuable tool for the characterization of solution properties of protein-ligand systems at the single molecule level, predicting relative off-rates, potentially of great value for combinatorial chemistry and biology.

Statistical significance of protein structure prediction by threading

In this study, we estimate the statistical significance of structure prediction by threading. We introduce a single parameter ɛ that serves as a universal measure determining the probability that the best alignment is indeed a native-like analog. Parameter ɛ takes into account both length and composition of the query sequence and the number of decoys in threading simulation. It can be computed directly from the query sequence and potential of interactions, eliminating the need for sequence reshuffling and realignment. Although our theoretical analysis is general, here we compare its predictions with the results of gapless threading. Finally we estimate the number of decoys from which the native structure can be found by existing potentials of interactions. We discuss how this analysis can be extended to determine the optimal gap penalties for any sequence-structure alignment (threading) method, thus optimizing it to maximum possible performance.

A single gene of chloroplast origin codes for mitochondrial and chloroplastic methionyl–tRNA synthetase in Arabidopsis thaliana

One-fifth of the tRNAs used in plant mitochondrial translation is coded for by chloroplast-derived tRNA genes. To understand how aminoacyl–tRNA synthetases have adapted to the presence of these tRNAs in mitochondria, we have cloned an Arabidopsis thaliana cDNA coding for a methionyl–tRNA synthetase. This enzyme was chosen because chloroplast-like elongator tRNAMet genes have been described in several plant species, including A. thaliana. We demonstrate here that the isolated cDNA codes for both the chloroplastic and the mitochondrial methionyl–tRNA synthetase (MetRS). The protein is transported into isolated chloroplasts and mitochondria and is processed to its mature form in both organelles. Transient expression assays using the green fluorescent protein demonstrated that the N-terminal region of the MetRS is sufficient to address the protein to both chloroplasts and mitochondria. Moreover, characterization of MetRS activities from mitochondria and chloroplasts of pea showed that only one MetRS activity exists in each organelle and that both are indistinguishable by their behavior on ion exchange and hydrophobic chromatographies. The high degree of sequence similarity between A. thaliana and Synechocystis MetRS strongly suggests that the A. thaliana MetRS gene described here is of chloroplast origin.

Protein component of the ribozyme ribonuclease P alters substrate recognition by directly contacting precursor tRNA

The protein component of ribonuclease P (RNase P) binds to the RNA subunit, forming a functional ribonucleoprotein complex in vivo and enhancing the affinity of the precursor tRNA (pre-tRNA) substrate. Photocrosslinking experiments with pre-tRNA bound to RNase P reconstituted with the protein component of Bacillus subtilis ribonuclease P (P protein) site specifically modified with a crosslinking reagent indicate that: (i) the central cleft of P protein directly interacts with the single-stranded 5′ leader sequence of pre-tRNA, and (ii) the orientation and register of the pre-tRNA leader sequence in the central cleft places the protein component in close proximity to the active site. This unique mode of interaction suggests that the catalytic active site in RNase P occurs near the interface of RNA and protein. In contrast to other ribonucleoprotein complexes where the protein mainly stabilizes the active tertiary fold of the RNA, a critical function of the protein component of RNase P is to alter substrate specificity and enhance catalytic efficiency.

A protein required for nuclear-protein import, Mog1p, directly interacts with GTP–Gsp1p, the Saccharomyces cerevisiae Ran homologue

We previously isolated 25 temperature-sensitive gsp1 alleles of Saccharomyces cerevisiae Ran homologue, each of which possesses amino acid changes that differ from each other. We report here isolation of three multicopy suppressors—PDE2, NTF2, and a gene designated MOG1—all of which rescued a growth defect of these gsp1 strains. The gsp1 suppression occurred even in the absence of GSP2, another S. cerevisiae GSP1-like gene. Previously, NTF2 was reported to suppress gsp1 but not PDE2. Mog1p, with a calculated molecular mass of 24 kDa, was found to be encoded by the yeast ORF YJR074W. Both MOG1 and NTF2 suppressed a series of gsp1 alleles with similar efficiency, and both suppressed gsp1 even with a single gene dose. Consistent with the high efficiency of gsp1 suppression, Mog1p directly bound to GTP, but not to GDP-Gsp1p. The disruption of MOG1 made yeast temperature-sensitive for growth. Δmog1, which was suppressed by overexpression of NTF2, was found to have a defect in both classic and nonclassic nuclear localization signal-dependent nuclear-protein imports, but not in mRNA export. Thus, Mog1p, which was localized in the nucleus, is a Gsp1p-binding protein involved in nuclear-protein import and that functionally interacts with Ntf2p. Furthermore, the finding that PDE2 suppressed both gsp1 and rna1–1 indicates that the Ran GTPase cycle is regulated by the Ras-cAMP pathway.

Bidirectional imprinting of a single gene: GNAS1 encodes maternally, paternally, and biallelically derived proteins

The GNAS1 gene encodes the α subunit of the guanine nucleotide-binding protein Gs, which couples signaling through peptide hormone receptors to cAMP generation. GNAS1 mutations underlie the hormone resistance syndrome pseudohypoparathyroidism type Ia (PHP-Ia), so the maternal inheritance displayed by PHP-Ia has raised suspicions that GNAS1 is imprinted. Despite this suggestion, in most tissues Gsα is biallelically encoded. In contrast, the large G protein XLαs, also encoded by GNAS1, is paternally derived. Because the inheritance of PHP-Ia predicts the existence of maternally, rather than paternally, expressed transcripts, we have investigated the allelic origin of other mRNAs derived from GNAS1. We find this gene to be remarkable in the complexity of its allele-specific regulation. Two upstream promoters, each associated with a large coding exon, lie only 11 kb apart, yet show opposite patterns of allele-specific methylation and monoallelic transcription. The more 5′ of these exons encodes the neuroendocrine secretory protein NESP55, which is expressed exclusively from the maternal allele. The NESP55 exon is 11 kb 5′ to the paternally expressed XLαs exon. The transcripts from these two promoters both splice onto GNAS1 exon 2, yet share no coding sequences. Despite their structural unrelatedness, the encoded proteins, of opposite allelic origin, both have been implicated in regulated secretion in neuroendocrine tissues. Remarkably, maternally (NESP55), paternally (XLαs), and biallelically (Gsα) derived proteins all are produced by different patterns of promoter use and alternative splicing of GNAS1, a gene showing simultaneous imprinting in both the paternal and maternal directions.

Computer-based analysis of the binding steps in protein complex formation

Computer models were used to examine whether and under what conditions the multimeric protein complex is inhibited by high concentrations of one of its components—an effect analogous to the prozone phenomenon in precipitin tests. A series of idealized simple “ball-and-stick” structures representing small oligomeric complexes of protein molecules formed by reversible binding reactions were analyzed to determine the binding steps leading to each structure. The equilibrium state of each system was then determined over a range of starting concentrations and Kds and the steady-state concentration of structurally complete oligomer calculated for each situation. A strong inhibitory effect at high concentrations was shown by any protein molecule forming a bridge between two or more separable parts of the complex. By contrast, proteins linked to the outside of the complex by a single bond showed no inhibition whatsoever at any concentration. Nonbridging, multivalent proteins in the body of the complex could show an inhibitory effect or not depending on the structure of the complex and the strength of its bonds. On the basis of this study, we suggest that the prozone phenomenon will occur widely in living cells and that it could be a crucial factor in the regulation of protein complex formation.

The interphotoreceptor retinoid binding protein gene in therian mammals: Implications for higher level relationships and evidence for loss of function in the marsupial mole

The subclass Theria of Mammalia includes marsupials (infraclass Metatheria) and placentals (infraclass Eutheria). Within each group, interordinal relationships remain unclear. One limitation of many studies is incomplete ordinal representation. Here, we analyze DNA sequences for part of exon 1 of the interphotoreceptor retinoid binding protein gene, including 10 that are newly reported, for representatives of all therian orders. Among placentals, the most robust clades are Cetartiodactyla, Paenungulata, and an expanded African clade that includes paenungulates, tubulidentates, and macroscelideans. Anagalida, Archonta, Altungulata, Hyracoidea + Perissodactyla, Ungulata, and the “flying primate” hypothesis are rejected by statistical tests. Among marsupials, the most robust clade includes all orders except Didelphimorphia. The phylogenetic placement of the monito del monte and the marsupial mole remains unclear. However, the marsupial mole sequence contains three frameshift indels and numerous stop codons in all three reading frames. Given that the interphotoreceptor retinoid binding protein gene is a single-copy gene that functions in the visual cycle and that the marsupial mole is blind with degenerate eyes, this finding suggests that phenotypic degeneration of the eyes is accompanied by parallel changes at the molecular level as a result of relaxed selective constraints.

Expression of sterol regulatory element-binding protein 1c (SREBP-1c) mRNA in rat hepatoma cells requires endogenous LXR ligands

The current paper describes a line of cultured rat hepatoma cells (McA-RH7777 cells) that mimics the behavior of rat liver by producing an excess of mRNA for sterol regulatory element-binding protein 1c (SREBP-1c) as opposed to SREBP-1a. These two transcripts are derived from a single gene by use of alternative promoters that are separated by many kilobases in the genome. The high level of SREBP-1c mRNA is abolished when cholesterol synthesis is blocked by compactin, an inhibitor of 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase that inhibits cholesterol synthesis. Levels of SREBP-1c mRNA are restored by mevalonate, the product of the HMG CoA reductase reaction, and by ligands for the nuclear hormone receptor LXR, including 22(R)-hydroxycholesterol and T0901317. These data suggest that transcription of the SREBP-1c gene in hepatocytes requires tonic activation of LXR by an oxysterol intermediate in the cholesterol biosynthetic pathway. Reduction of this intermediate lowers SREBP-1c levels, and this in turn is predicted to lower the rates of fatty acid biosynthesis in liver.