Architecture of the RNA polymerase II-Mediator core initiation complex.

The conserved co-activator complex Mediator enables regulated transcription initiation by RNA polymerase (Pol) II. Here we reconstitute an active 15-subunit core Mediator (cMed) comprising all essential Mediator subunits from Saccharomyces cerevisiae. The cryo-electron microscopic structure of cMed bound to a core initiation complex was determined at 9.7 Å resolution. cMed binds Pol II around the Rpb4-Rpb7 stalk near the carboxy-terminal domain (CTD). The Mediator head module binds the Pol II dock and the TFIIB ribbon and stabilizes the initiation complex. The Mediator middle module extends to the Pol II foot with a 'plank' that may influence polymerase conformation. The Mediator subunit Med14 forms a 'beam' between the head and middle modules and connects to the tail module that is predicted to bind transcription activators located on upstream DNA. The Mediator 'arm' and 'hook' domains contribute to a 'cradle' that may position the CTD and TFIIH kinase to stimulate Pol II phosphorylation.

Cryo-electron tomography: methodology, developments and biological applications.

Cryo-electron tomography allows three-dimensional visualization of frozen-hydrated, vitrified biological material at molecular resolution. Here, we summarize the most important sample preparation methods and technical aspects relevant for cryo-electron tomography, as well as its recent biological applications from isolated macromolecular complexes to entire cells and tissues.

A tale of two giant proteases.

The 26S proteasome and tripeptidyl peptidase II (TPPII) are two exceptionally large eukaryotic protein complexes involved in intracellular proteolysis, where they exert their function sequentially: the proteasome, a multisubunit complex of 2.5 MDa, acts at the downstream end of the ubiquitin pathway and degrades ubiquitinylated proteins into small oligopeptides. Such oligopeptides are substrates for TPPII, a 6-MDa homooligomer, which releases tripeptides from their free N-terminus. Both 26S and TPPII are very fragile complexes refractory to crystallization and in their fully assembled native form have been visualized only by electron microscopy. Here, we will discuss the structural features of the two complexes and their functional implications.

The Chaperones of the archaeon Thermoplasma acidophilum.

Chaperonesare an essential component of a cell's ability to respond to environmental challenges. Chaperones have been studied primarily in bacteria, but in recent years it has become apparent that some classes of chaperones either are very divergent in bacteria relative to archaea and eukaryotes or are missing entirely. In contrast, a high degree of similarity was found between the chaperonins of archaea and those of the eukaryotic cytosol, which has led to the establishment of archaeal model systems. The archaeon most extensively used for such studies is Thermoplasma acidophilum, which thrives at 59 degrees C and pH 2. Here we review information on its chaperone complement in light of the recently determined genome sequence.

ATP-induced structural change of the thermosome is temperature-dependent.

Protein folding by chaperonins is powered by ATP binding and hydrolysis. ATPase activity drives the folding machine through a series of conformational rearrangements, extensively described for the group I chaperonin GroEL from Escherichia coli but still poorly understood for the group II chaperonins. The latter--archaeal thermosome and eukaryotic TRiC/CCT--function independently of a GroES-like cochaperonin and are proposed to rely on protrusions of their own apical domains for opening and closure in an ATP-controlled fashion. Here we use small-angle neutron scattering to analyze structural changes of the recombinant alpha-only and the native alphabeta-thermosome from Thermoplasma acidophilum upon their ATPase cycling in solution. We show that specific high-salt conditions, but not the presence of MgATP alone, induce formation of higher order thermosome aggregates. The mechanism of the open-closed transition of the thermosome is strongly temperature-dependent. ATP binding to the chaperonin appears to be a two-step process: at lower temperatures an open state of the ATP-thermosome is predominant, whereas heating to physiological temperatures induces its switching to a closed state. Our data reveal an analogy between the ATPase cycles of the two groups of chaperonins and enable us to put forward a model of thermosome action.

FhuA-mediated phage genome transfer into liposomes: a cryo-electron tomography study.

BACKGROUND: The transfer of phage genomes into host cells is a well established but only dimly understood process. Following the irreversible phage binding to a receptor in the bacterial outer membrane, the DNA is ejected from the viral capsid and transferred across the bacterial cell envelope. In Escherichia coli, the mere interaction of the phage T5 with its outer membrane receptor, the ferrichrome transporter FhuA, is sufficient to trigger the release of the DNA from the phage capsid. Although the structure of FhuA has been determined at atomic resolution, the understanding of the respective roles of phage and bacterial proteins in DNA channeling and the mechanisms by which the transfer of the DNA is mediated remains fragmentary. RESULTS: We report on the use of cryo-electron tomography to analyze, at a molecular level, the interactions of T5 phages bound to FhuA-containing proteoliposomes. The resolution of the three-dimensional reconstructions allowed us to visualize the phage-proteoliposome interaction before and after release of the genome into the vesicles. After binding to its receptor, the straight fiber of the phage T5 (the "tip" of the viral tail made of pb2 proteins) traverses the lipid bilayer, allowing the transfer of its double-stranded DNA (121,000 bp) into the proteoliposome. Concomitantly, the tip of the tail undergoes a major conformational change; it shrinks in length (from 50 to 23 nm), while its diameter increases (from 2 to 4 nm). CONCLUSIONS: Taking into account the crystal structure of FhuA, we conclude that FhuA is only used as a docking site for the phage. The tip of the phage tail acts like an "injection needle," creating a passageway at the periphery of FhuA, through which the DNA crosses the membrane. A possible mechanistic scenario for the transfer of the viral genome into bacteria is discussed.

Purification, crystallization, and preliminary X-ray diffraction analysis of the Tricorn protease hexamer from Thermoplasma acidophilum.

Tricorn protease from Thermoplasma acidophilum is a hexameric enzyme; in vivo the hexamers assemble further to form large icosahedral capsids of 14.6 MDa. Recombinant Tricorn protease was purified as an enzymatically active hexamer of 0.72 MDa that formed crystals of octahedral morphology under low-ionic-strength conditions. These crystals belong to space group C2 with unit cell dimensions a = 307.5 A, b = 163.2 A, c = 220.9 A, beta = 105.5 degrees and diffract to 2.2-A resolution using high-brilliance synchrotron radiation. Based on analysis of the self-rotation function and the presence of a pseudo-origin peak in the native Patterson map, a packing model was derived for the complex, comprising 1.5 hexamers per asymmetric unit with a solvent content of 43%. Due to the ninefold noncrystallographic symmetry the Tricorn crystals represent an interesting case for phasing X-ray crystallographic data by electron microscopic phase information.

Tricorn protease in bacteria: characterization of the enzyme from Streptomyces coelicolor.

Tricorn protease is believed to act downstream of the proteasome, or of other ATP-dependent proteases, cleaving the oligopeptides (mostly 6 to 12 residues) released by them into small peptides (2 to 4 residues), before an array of aminopeptidases finally converts them into free amino acids. Hitherto, the occurrence of Tricorn protease seemed to be limited to some archaea, but genes encoding Tricorn homologs have now been found in several bacterial genomes. Among them is Streptomyces coelicolor A3(2), which has, in fact, two Tricorn-like genes, ScC77.16c and ScE87.19. The proteins encoded by them (TRI-ScC77 and TRI-ScE87) are very similar in their PDZ and TSP domains, but rather divergent in their beta-propeller domains. We have expressed one of them, TRI-ScC77, in E. coil and characterized the recombinant protein structurally and functionally. TRI-ScC77 forms a homohexameric complex of approximately 700 kDa, both in E. coil and in S. coelicolor, with enzymatic properties very similar to the complex from the archaeon Thermoplasma acidophilum. The fact that Tricorn-like proteins exist not only in thermoacidophiles, but also in bacteria inhabiting radically different environments, rules out the possibility that Tricorn protease is an adaptive element that helps to meet the challenges of an extreme habitat.

Macromolecular electron microscopy in the era of structural genomics.

Macromolecular machines carry out many cellular functions. Cryo-electron microscopy (cryo-EM) is emerging as a powerful method for studying the structure, assembly and dynamics of such macromolecules, and their interactions with substrates. With resolutions still improving, 'single-particle' analyses are already depicting secondary structure. Moreover, cryo-EM can be combined in several ways with X-ray diffraction to enhance the resolution of cryo-EM and the applicability of crystallography. Electron tomography holds promise for visualizing machines at work inside cells.

Toward detecting and identifying macromolecules in a cellular context: template matching applied to electron tomograms.

Electron tomography is the only technique available that allows us to visualize the three-dimensional structure of unfixed and unstained cells currently with a resolution of 6-8 nm, but with the prospect to reach 2-4 nm. This raises the possibility of detecting and identifying specific macromolecular complexes within their cellular context by virtue of their structural signature. Templates derived from the high-resolution structure of the molecule under scrutiny are used to search the reconstructed volume. Here we outline and test a computationally feasible two-step procedure: In a first step, mean-curvature motion is used for segmentation, yielding subvolumes that contain with a high probability macromolecules in the expected size range. Subsequently, the particles contained in the subvolumes are identified by cross-correlation, using a set of three-dimensional templates. With simulated and real tomographic data we demonstrate that such an approach is feasible and we explore the detection limits. Even structurally similar particles, such as the thermosome, GroEL, and the 20S proteasome can be identified with high fidelity. This opens up exciting prospects for mapping the territorial distribution of macromolecules and for analyzing molecular interactions in situ.

Intervesicle cross-linking with integrin alpha IIb beta 3 and cyclic-RGD-lipopeptide. A model of cell-adhesion processes.

We report the synthesis of a new integrin alpha(IIb)beta(3)-specific cyclic hexapeptide that contains an Arg-Gly-Asp (RGD) sequence and is coupled to a dimyristoylthioglyceryl anchor. We demonstrate that this ligand is useful to study specific integrin binding to membrane surfaces. With the help of biotinylated analogues of the peptide, a spacer of optimal length between the peptide and lipid moieties was searched for by evaluating the binding strength with an enzyme-coupled immunosorbent assay (ELISA) and by surface plasmon resonance (SPR). It was found to be strongly dependent on the length of the spacer introduced between the biotin and peptide moieties of the ligands, which consisted either of epsilon-aminohexanoic acid (epsilonAhx) or of epsilonAhx with two additional glycine units. Best results were obtained with c[Arg-Gly-Asp-D-Phe-Lys(Biot-Ahx-Gly-Gly)-Gly-] with dissociation constants of K(D) = 0.158 microM from ELISA and K(D) = 1.1 microM from SPR measurements. The analogous lipopeptide, c[Arg-Gly-Asp-D-Phe-Lys([dimyristoyl-3-thioglyceryl-succinimido -propanoyl]Ahx-Gly-Gly)-Gly], was used as a membrane-anchored integrin ligand. It is shown by fluorescence microscopy and cryo electron microscopy that integrin reconstituted into phospholipid vesicles binds to vesicles decorated with the lipopeptide, forming regularly spaced bridges between the two kinds of vesicles. The novel integrin-specific ligand allows establishment of new model systems for systematic studies of the self-organization of integrin clusters and focal adhesion complexes.

The genome sequence of the thermoacidophilic scavenger Thermoplasma acidophilum.

Thermoplasma acidophilum is a thermoacidophilic archaeon that thrives at 59 degrees C and pH 2, which was isolated from self-heating coal refuse piles and solfatara fields. Species of the genus Thermoplasma do not possess a rigid cell wall, but are only delimited by a plasma membrane. Many macromolecular assemblies from Thermoplasma, primarily proteases and chaperones, have been pivotal in elucidating the structure and function of their more complex eukaryotic homologues. Our interest in protein folding and degradation led us to seek a more complete representation of the proteins involved in these pathways by determining the genome sequence of the organism. Here we have sequenced the 1,564,905-base-pair genome in just 7,855 sequencing reactions by using a new strategy. The 1,509 open reading frames identify Thermoplasma as a typical euryarchaeon with a substantial complement of bacteria-related genes; however, evidence indicates that there has been much lateral gene transfer between Thermoplasma and Sulfolobus solfataricus, a phylogenetically distant crenarchaeon inhabiting the same environment. At least 252 open reading frames, including a complete protein degradation pathway and various transport proteins, resemble Sulfolobus proteins most closely.

Crystal structure of the beta-apical domain of the thermosome reveals structural plasticity in the protrusion region.

The crystal structure of the beta-apical domain of the thermosome, an archaeal group II chaperonin from Thermoplasma acidophilum, has been determined at 2.8 A resolution. The structure shows an invariant globular core from which a 25 A long protrusion emanates, composed of an elongated alpha-helix (H10) and a long extended stretch consisting of residues GluB245-ThrB253. A comparison with previous apical domain structures reveals a large segmental displacement of the protruding part of helix H10 via the hinge GluB276-ValB278. The region comprising residues GluB245-ThrB253 adopts an extended beta-like conformation rather than the alpha-helix seen in the alpha-apical domain. Consequently, it appears that the protrusions of the apical domains from group II chaperonins might assume a variety of context-dependent conformations during an open, substrate-accepting state of the chaperonin. Sequence variations in the protrusion regions that are found in the eukaryotic TRiC/CCT subunits may provide different structural propensities and hence serve different roles in substrate recognition.

ATPase cycle controls the conformation of an archaeal chaperonin as visualized by cryo-electron microscopy.

Chaperonins are double-ring protein folding machines fueled by ATP binding and hydrolysis. Conformational rearrangements upon ATPase cycling of the group I chaperonins, typified by the Escherichia coli GroEL/GroES system, have been thoroughly investigated by cryo-electron microscopy and X-ray crystallography. For archaeal group II chaperonins, however, these methods have so far failed to provide a correlation between the structural and the functional states. Here, we show that the conformation of the native alphabeta-thermosome of Thermoplasma acidophilum in vitrified ice is strictly regulated by adenine nucleotides.

The regulatory complex of Drosophila melanogaster 26S proteasomes. Subunit composition and localization of a deubiquitylating enzyme.

Drosophila melanogaster embryos are a source for homogeneous and stable 26S proteasomes suitable for structural studies. For biochemical characterization, purified 26S proteasomes were resolved by two-dimensional (2D) gel electrophoresis and subunits composing the regulatory complex (RC) were identified by amino acid sequencing and immunoblotting, before corresponding cDNAs were sequenced. 17 subunits from Drosophila RCs were found to have homologues in the yeast and human RCs. An additional subunit, p37A, not yet described in RCs of other organisms, is a member of the ubiquitin COOH-terminal hydrolase family (UCH). Analysis of EM images of 26S proteasomes-UCH-inhibitor complexes allowed for the first time to localize one of the RC's specific functions, deubiquitylating activity. The masses of 26S proteasomes with either one or two attached RCs were determined by scanning transmission EM (STEM), yielding a mass of 894 kD for a single RC. This value is in good agreement with the summed masses of the 18 identified RC subunits (932 kD), indicating that the number of subunits is complete.

ATPase cycle of an archaeal chaperonin.

Recent structural data imply differences in allosteric behavior of the group I chaperonins, typified by GroEL from Escherichia coli, and the group II chaperonins, which comprise archaeal thermosome and eukaryotic TRiC/CCT. Therefore, this study addresses the mechanism of interaction of adenine nucleotides with recombinant alpha-only and native alphabeta-thermosomes from Thermoplasma acidophilum, which also enables us to analyze the role of the heterooligomeric composition of the natural thermosome. Although all subunits of the alpha-only thermosome seem to bind nucleotides tightly and independently, the native chaperonin has two different classes of ATP-binding sites. Furthermore, for the alpha-only thermosome, the steady-state ATPase rate is determined by the cleavage reaction itself, whereas, for the alphabeta-thermosome, the rate-limiting step is associated with a post-hydrolysis isomerisation into a non-covalent ADP*P(i) species prior to the release of the gamma-phosphate group. After half-saturation with ATP, a negative cooperativity in hydrolysis is observed for both thermosomes. The effect of Mg(2+) and K(+) nucleotide cycling is documented. We conclude that archaeal chaperonins have unique allosteric properties and discuss them in the light of the mechanism established for the group I chaperonins.

Dis-assembly lines: the proteasome and related ATPase-assisted proteases.

Self-compartmentalizing proteases, such as the proteasome and several prokaryotic energy-dependent proteases, are designed to act in the crowded environment of the cell. Proteins destined for degradation are recognized and unfolded by regulatory subcomplexes that invariably contain ATPase modules, before being translocated into another subcomplex, the proteolytic core, for degradation. The sequential actions effected on substrates are reflected in the linear arrangement of these subcomplexes; thus, the holocomplexes are organized as molecular disassembly and degradation lines.

Cryo-electron tomography of neurospora mitochondria.

Cryo-electron tomography was used to study the structural organization of whole frozen-hydrated mitochondria from Neurospora crassa. Unlike mitochondria from many other species and tissues, in this case the cristae form a three-dimensional network of interconnected lamellae. Basically, the three-dimensional structure of ice-embedded mitochondria from this species is consistent with previous descriptions of mitochondria prepared by chemical fixation and resin embedding. Nonetheless, ice-embedded mitochondria display some important differences: the outer surface of the mitochondria was found to be rather smooth, the intermembrane space was constant in width, and distinct contact sites between the membranes were clearly revealed. Furthermore ATP synthase particles on the outer surface of an "inside-out vesicle" were visible in 3-D reconstructions. Thus, cryo-electron tomography can provide detailed insights into these organelles with minimal perturbations of the physiological state. This indicates that it is a realistic goal to achieve "molecular resolution" with rather large biological specimens in the near future, ultimately allowing the identification and localization of macromolecules in their cellular context.