Cooperativity in macromolecular assembly

The thermodynamic principle of cooperativity is used to drive the formation of specific macromolecular complexes during the assembly of a macromolecular machine. Understanding cooperativity provides insight into the mechanisms that govern assembly and disassembly of multicomponent complexes. Our understanding of assembly mechanisms is lagging considerably behind our understanding of the structure and function of these complexes. A significant challenge remains in tackling the thermodynamics and kinetics of the intermolecular interactions required for all cellular functions.     

Cartwheel assembly

The cartwheel is a subcentriolar structure consisting of a central hub and nine radially arranged spokes, located at the proximal end of the centriole. It appears at the initial stage of the centriole assembly process as the first ninefold symmetrical structure. The cartwheel was first described more than 50 years ago, but it is only recently that its pivotal role in establishing the ninefold symmetry of the centriole was demonstrated. Significant progress has since been made in understanding its fine structure and assembly mechanism. Most importantly, the central part of the cartwheel, from which the ninefold symmetry originates, is shown to form by self-association of nine dimers of the protein SAS-6. This finding, together with emerging data on other components of the cartwheel, has opened new avenues in centrosome biology.     

Virus assembly

Virus assembly is a term describing several areas of current research: protein-RNA recognition; the control of the formation of large complexes; and mechanisms of particle maturation. Our understanding of these processes is increasing as a result of the efforts of numerous studies. Crystal structures have recently been solved for relatively complex assembly intermediates.     

Metallocenter assembly in nickel-containing enzymes

 The five known nickel-dependent enzymes include urease, hydrogenase, carbon monoxide dehydrogenase (and CO dehydrogenase/acetyl-coenzyme A synthase), methyl-S–coenzyme M reductase, and one class of superoxide dismutase. Consistent with their disparate functions, these Ni enzymes have distinct metallocenter structures that vary in Ni coordination geometry, number and types of metals, and the presence of additional components. Sophisticated cellular Ni processing systems have been devised to allow for specific and functional incorporation of Ni into these proteins. This review highlights several themes that are common to the enzyme activation processes and summarizes current concepts related to the enzyme-specific Ni assembly pathways. Received, accepted: 3 April 1997     

Primordial follicular assembly in humans--revisited

Recent interest in the initial phases of ovarian follicular formation and development has lead to a number of publications in this area, most of which address the autocrine and paracrine factors involved in primordial follicle activation to primary follicle. Primordial follicle assembly (first step in follicle formation) determines the lifetime supply of primordial follicles and remains a poorly understood phenomenon. Despite a number of recent articles that are concentrating on immuno-histochemistry, basic steps in the process are not clear. Hence, we feel it is time to take a step back and see what is available in the literature and identify the gaps in which future research about primordial follicle assembly in humans needs to be directed.     

Visual circuit assembly in Drosophila

Both insect and vertebrate visual circuits are organized into orderly arrays of columnar and layered synaptic units that correspond to the array of photoreceptors in the eye. Recent genetic studies in Drosophila have yielded insights into the molecular and cellular mechanisms that pattern the layers and columns and establish specific connections within the synaptic units. A sequence of inductive events and complex cellular interactions coordinates the assembly of visual circuits. Photoreceptor-derived ligands, such as hedgehog and Jelly-Belly, induce target development and expression of specific adhesion molecules, which in turn serve as guidance cues for photoreceptor axons. Afferents are directed to specific layers by adhesive afferent-target interactions mediated by leucine-rich repeat proteins and cadherins, which are restricted spatially and/or modulated dynamically. Afferents are restricted to their topographically appropriate columns by repulsive interactions between afferents and by autocrine activin signaling. Finally, Dscam-mediated repulsive interactions between target neuron dendrites ensure appropriate combinations of postsynaptic elements at synapses. Essentially, all these Drosophila molecules have vertebrate homologs, some of which are known to carry out analogous functions. Thus, the studies of Drosophila visual circuit development would shed light on neural circuit assembly in general.     

Pattern formation in centrosome assembly

A striking but poorly explained feature of cell division is the ability to assemble and maintain organelles not bounded by membranes, from freely diffusing components in the cytosol. This process is driven by information transfer across biological scales such that interactions at the molecular scale allow pattern formation at the scale of the organelle. One important example of such an organelle is the centrosome, which is the main microtubule organising centre in the cell. Centrosomes consist of two centrioles surrounded by a cloud of proteins termed the pericentriolar material (PCM). Profound structural and proteomic transitions occur in the centrosome during specific cell cycle stages, underlying events such as centrosome maturation during mitosis, in which the PCM increases in size and microtubule nucleating capacity. Here we use recent insights into the spatio-temporal behaviour of key regulators of centrosomal maturation, including Polo-like kinase 1, CDK5RAP2 and Aurora-A, to propose a model for the assembly and maintenance of the PCM through the mobility and local interactions of its constituent proteins. We argue that PCM structure emerges as a pattern from decentralised self-organisation through a reaction-diffusion mechanism, with or without an underlying template, rather than being assembled from a central structural template alone. Self-organisation of this kind may have broad implications for the maintenance of mitotic structures, which, like the centrosome, exist stably as supramolecular assemblies on the micron scale, based on molecular interactions at the nanometer scale.     

Intermediates in adenovirus assembly.

Three intermediates in adenovirus assembly have been defined; nuclear intermediates, young virions, and mature virions. The nuclear intermediates are fragile and heterogenous in size (550S-670S) and withstand separation on ficoll gradients but fall apart upon CsCl gradient centrifugation unless prefixed with glutaraldehyde. They contain both capsid and core structures, and the core structures are preferentially released during purification in CsCl. The precursor polypeptides pVI and pVII are present in the intermediates without any corresponding mature polypeptide. The young virions (Ishibashi and Maizel, 1974) are stable and preferentially confined to the nuclei after cell fractionation. They contain both uncleaved precursor polypeptides and their cleavage products. The mature virions accumulate in the cytoplasm during cell fractionation and contain the final mature polypeptides. Pulse-chase labeling kinetics, focusing on the precursor polypeptides, suggest that these three classes participate in assembly of adenovirus. Tryptic peptide maps establish that polypeptide pVI is the precursor of polypeptide VI, but only a small fraction of polypeptide 26K can in vivo account for polypeptide VIII.     

Flagellar assembly in Salmonella typhimurium

The bacterial flagellum is a motility apparatus in which a long helical filament - the propeller - is driven by a rotary motor embedded in the cell surface. Out of more than 40 genes required for construction of a fully functional flagellum in Salmonella typhimurium, only 18 gene products have been identified in the mature structure. Some other flagellar proteins play logistical roles during construction, which involves the selective export of flagellar components through a central hole in the flagellum. The whole structure is constructed from base to tip by linear assembly; that is, by adding new components on the growing end, resulting in the distal growth of each substructure. Components of the substructures do not necessarily self-assemble, but often demand the help of other proteins. Recent progress in the understanding of flagellar assembly, which has been most extensively studied in S. typhimurium, is reviewed.     

Laminins in basement membrane assembly

The heterotrimeric laminins are a defining component of all basement membranes and self-assemble into a cell-associated network. The three short arms of the cross-shaped laminin molecule form the network nodes, with a strict requirement for one α, one β and one γ arm. The globular domain at the end of the long arm binds to cellular receptors, including integrins, α-dystroglycan, heparan sulfates and sulfated glycolipids. Collateral anchorage of the laminin network is provided by the proteoglycans perlecan and agrin. A second network is then formed by type IV collagen, which interacts with the laminin network through the heparan sulfate chains of perlecan and agrin and additional linkage by nidogen. This maturation of basement membranes becomes essential at later stages of embryo development.     

合成生物学中的DNA组装技术

合成生物学旨在应用工程学的研究思路及手段去设计或改造生物系统,是一个综合了科学与工程的拥有发展潜力的新兴学科,在生物医药、农业、能源、环保等方面发挥着巨大作用。DNA组装技术是合成生物学中的关键技术,也是合成生物学快速发展的限制性技术。综述了众多DNA组装技术的发展及其在合成生物学研究中的意义和应用。     

Species assembly in model ecosystems, II: Results of the assembly process

In the companion paper of this set (Capitán and Cuesta, 2010) we have developed a full analytical treatment of the model of species assembly introduced in Capitán et al. (2009). This model is based on the construction of an assembly graph containing all viable configurations of the community, and the definition of a Markov chain whose transitions are the transformations of communities by new species invasions. In the present paper we provide an exhaustive numerical analysis of the model, describing the average time to the recurrent state, the statistics of avalanches, and the dependence of the results on the amount of available resource. Our results are based on the fact that the Markov chain provides an asymptotic probability distribution for the recurrent states, which can be used to obtain averages of observables as well as the time variation of these magnitudes during succession, in an exact manner. Since the absorption times into the recurrent set are found to be comparable to the size of the system, the end state is quickly reached (in units of the invasion time). Thus, the final ecosystem can be regarded as a fluctuating complex system where species are continually replaced by newcomers without ever leaving the set of recurrent patterns. The assembly graph is dominated by pathways in which most invasions are accepted, triggering small extinction avalanches. Through the assembly process, communities become less resilient (e.g., have a higher return time to equilibrium) but become more robust in terms of resistance against new invasions.     

Mitochondrial ribosome assembly in Neurospora crassa: mutants with defects in mitochondrial ribosome assembly.

Summary We have examined mitochondrial (mt) ribosome assembly and-function in five nuclear and six extranuclear mutants of Neurospora crassa which had previously been characterized as deficient in cytochromes b and aa ₃. All six extranuclear mutants showed phenotypes similar to that previously described for the extranuclear [poky] mutant: small subunit-deficient with 19 S rRNA rapidly degraded. The nuclear mutants have the following phenotypes: 297-24 is mt small subunit deficient with 19 S RNA rapidly degraded. 289-56 is mt small subunit deficient but contains normal ratios of 19 S to 25 S RNA in whole mitochondria. 289-67 and 299-9 show defects in the processing of 25 S RNA leading to accumulation of a large precursor RNA. 289-4 is deficient in large subunits although a substantial, but less than normal, amount of 25 S RNA is present in the mitochondria.The present work provides new insight into the phenotypes of mt small subunit-deficient mutants. Previous studies using chloramphenicol suggest that some defects in the assembly of mt small subunits may arise secondarily as a result of inhibition of mt protein synthesis (LaPolla and Lambowitz, 1977; Lambowitz et al., 1979). Three mutants (289-56, 289-67 and 299-9) appear to show such defects. These strains contain incomplete mt small subunits which sediment more slowly than normal and are deficient in at least two proteins, S-5 and S-9. Correlation of mutant phenotypes with rates of mt protein synthesis in the different strains suggests that mt protein synthesis must be decreased to less than one half of the wild-type rate before secondary defects in mt small subunit assembly are observed. This threshold value is much lower than that which leads to gross deficiencies of cytochromes b and aa ₃. Although several mutants have phenotypes suggestive of alterations in mt ribosomal proteins, no such alterations could be identified by two dimensional gel electrophoresis.     

Combinatorial metabolic pathway assembly approaches and toolkits for modular assembly

Synthetic Biology is a rapidly growing interdisciplinary field that is primarily built upon foundational advances in molecular biology combined with engineering design principles such as modularity and interoperability. The field considers living systems as programmable at the genetic level and has been defined by the development of new platform technologies and methodological advances. A key concept driving the field is the Design-Build-Test-Learn cycle which provides a systematic framework for building new biological systems. One major application area for synthetic biology is biosynthetic pathway engineering that requires the modular assembly of different genetic regulatory elements and biosynthetic enzymes. In this review we provide an overview of modular DNA assembly and describe and compare the plethora of in vitro and in vivo assembly methods for combinatorial pathway engineering. Considerations for part design and methods for enzyme balancing are also presented, and we briefly discuss alternatives to intracellular pathway assembly including microbial consortia and cell-free systems for biosynthesis. Finally, we describe computational tools and automation for pathway design and assembly and argue that a deeper understanding of the many different variables of genetic design, pathway regulation and cellular metabolism will allow more predictive pathway design and engineering.     

Human immunodeficiency virus type 1 Gag assembly through assembly intermediates

Human immunodeficiency virus Gag protein self-assembles into spherical particles, and recent reports suggest the formation of assembly intermediates during the process. To understand the nature of such assembly intermediates along with the mechanism of Gag assembly, we employed expression in Escherichia coli and an in vitro assembly reaction. When E. coli expression was performed at 37 degrees C, Gag predominantly assembled to a high order of multimer, apparently equivalent to the virus-like particles obtained following Gag expression in eukaryotic cells, through the formation of low orders of multimer characterized with a discreet sedimentation value of 60 S. Electron microscopy confirmed the presence of spherical particles in the E. coli cells. In contrast, expression at 30 degrees C resulted in the production of only the 60 S form of Gag multimer, and crescent-shaped structures or small patches with double electron-dense layers were accumulated, but no complete particles. In vitro assembly reactions using purified Gag protein, when performed at 37 degrees C, also produced the high order of Gag multimers with some 60 S multimers, whereas the 30 degrees C reaction produced only the 60 S multimers. However, when the 60 S multimers were cross-linked so as not to allow conformational changes, in vitro assembly reactions at 37 degrees C did not produce any higher order of multimers. ATP depletion did not halt Gag assembly in the E. coli cells, and the addition of GroEL-GroES to in vitro reactions did not facilitate Gag assembly, indicating that conformational changes rather than protein refolding by chaperonins, induced at 37 degrees C, were solely responsible for the Gag assembly observed here. We suggest that Gag assembles to a capsid through the formation of the 60 S multimer, possibly a key intermediate of the assembly process, accompanied with conformational changes in Gag.