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PubMed1996

Assembly of the phagocyte NADPH oxidase: molecular interaction of oxidase proteins.

Phagocytes such as neutrophils play a key role in the body's innate immune response to infection. These cells travel throughout the body in search of pathogens and are rapidly mobilized to sites of inflammation where they phagocytose these pathogens and subsequently release a variety of toxic oxygen radical species and proteolytic enzymes to directly destroy the engulfed particle. The generation of microbicidal oxidants by neutrophils results from the action of a multi-protein enzymatic complex known as the NADPH oxidase. Altogether, there are currently seven proteins reported to be associated with the NADPH oxidase assembly. In resting neutrophils, these NADPH oxidase protein components are segregated into cytoplasmic and plasma membrane compartments. However, during assembly and activation of the NADPH oxidase, the cytosolic protein components translocate to the plasma membrane or phagosomal membrane where they assemble around a central membrane-bound protein known as flavocytochrome b. This assembly process is highly regulated and involves multiple binding interactions between the individual NADPH oxidase proteins, resulting in an active oxidase complex. Over the past few years, a number of these sites of binding interaction between the oxidase proteins have been identified, leading to a clearer understanding of the intermolecular interactions occurring among protein components during the assembly process. In addition, this information has contributed to our understanding of the roles played by each protein during the activation and assembly process. In this review, we describe the key features of each NADPH oxidase protein and then summarize our current understanding of the specific molecular interactions occurring between these proteins, focusing on the role these protein:protein binding interactions play in the NADPH oxidase assembly process.

Bacterial Proteins↗

PubMed2006

Effect of impact assembly on the fretting corrosion of modular hip tapers.

The goal of this study was to determine the effect of assembly load and local assembly environmental conditions on the fretting corrosion of modular femoral stem tapers. Femoral head/taper assemblies in both similar (CoCrMo/CoCrMo) and mixed (CoCrMo/Ti-6Al-4V) alloy combinations were evaluated using an electrochemical test method. Specimens were assembled under impact loading and by hand, in both wet and dry conditions. Incremental cyclic loads ranging from 89 to 5,340 N were applied at a frequency of 3 Hz in Ringer's solution at ambient temperature. During the test, both the open circuit potential (OCP) and fretting current (i(fret)) were measured using a saturated calomel electrode (SCE) and counter electrode, respectively. The results were comparable for both mixed and similar alloy couples. Decreases in OCP and increases in i(fret) (indicators of oxide film fracture and repassivation) were seen with increasing load magnitude, often occurring at loads well below those expected clinically. OCP at the 5,340 N cyclic load ranged from -30.4 to -103.7 mV versus SCE for similar alloy couples, and -19.1 to -181.4 mV versus SCE for mixed alloy couples. Mean peak fretting currents ranged from 0.84 to 1.42 microA and 1.06 to 3.12 microA for similar and mixed alloy couples, respectively. The larger current magnitudes and more negative shifts in OCP for mixed alloy couples indicate the difference in oxide film fracture behavior between titanium and cobalt alloys. The load at which OCP began to drop (onset of fretting) was dependent upon the assembly conditions for both material couples. Specimens assembled with impact loads in air showed the highest resistance to fretting. The results of this study indicate that the assembly load and the environment both play a role in the initial stability of modular hip taper connections.

Chromium Alloys↗

PubMed1998

Involvement of two novel chaperones in the assembly of mitochondrial NADH:Ubiquinone oxidoreductase (complex I).

The respiratory complex I of mitochondria consists of some 40 different subunits which form an L-shaped structure. Perpendicular to a hydrophobic arm embedded in the inner mitochondrial membrane a peripheral arm protrudes into the matrix. Assembly of the complex as studied in the fungus Neurospora crassa involves the formation of discrete intermediates. The matrix arm and the membrane arm are formed independently of each other and are joined in the course of assembly. The membrane arm itself is formed by association of two assembly intermediates, a smaller of 200 kDa and a larger of 350 kDa. The latter is associated with two extra proteins of 84 and 30 kDa which are not constituent parts of mature complex I. Their primary structures show no similarity to known proteins. Mutants generated by disrupting the genes of either of the two proteins accumulate the matrix arm of complex I and the small membrane arm assembly intermediate, but are incapable of forming the large intermediate. In the wild-type, the extra proteins exclusively associate with the large membrane arm assembly intermediate. Pulse-chase labelling experiments showed that the two proteins are repeatedly involved in many assembly cycles of the intermediate. These results indicate that the two proteins are novel chaperones specific for complex I membrane arm assembly.

Amino Acid Sequence↗

PubMed2000

Small heat-shock protein structures reveal a continuum from symmetric to variable assemblies.

The small heat-shock proteins (sHSPs) form a diverse family of proteins that are produced in all organisms. They function as chaperone-like proteins in that they bind unfolded polypeptides and prevent uncontrolled protein aggregation. Here, we present parallel cryo-electron microscopy studies of five different sHSP assemblies: Methanococcus jannaschii HSP16.5, human alphaB-crystallin, human HSP27, bovine native alpha-crystallin, and the complex of alphaB-crystallin and unfolded alpha-lactalbumin. Gel-filtration chromatography indicated that HSP16.5 is the most monodisperse, while HSP27 and the alpha-crystallin assemblies are more polydisperse. Particle images revealed a similar trend showing mostly regular and symmetric assemblies for HSP16.5 particles and the most irregular assemblies with a wide range of diameters for HSP27. A symmetry test on the particle images indicated stronger octahedral symmetry for HSP16.5 than for HSP27 or the alpha-crystallin assemblies. A single particle reconstruction of HSP16.5, based on 5772 particle images with imposed octahedral symmetry, resulted in a structure that closely matched the crystal structure. In addition, the cryo-EM reconstruction revealed internal density presumably corresponding to the flexible 32 N-terminal residues that were not observed in the crystal structure. The N termini were found to partially fill the central cavity making it unlikely that HSP16.5 sequesters denatured proteins in the cavity. A reconstruction calculated without imposed symmetry confirmed the presence of at least loose octahedral symmetry for HSP16.5 in contrast to the other sHSPs examined, which displayed no clear overall symmetry. Asymmetric reconstructions for the alpha-crystallin assemblies, with an additional mass selection step during image processing, resulted in lower resolution structures. We interpret the alpha-crystallin reconstructions to be average representations of variable assemblies and suggest that the resolutions achieved indicate the degree of variability. Quaternary structural information derived from cryo-electron microscopy is related to recent EPR studies of the alpha-crystallin domain fold and dimer interface of alphaA-crystallin.

Animals↗

PubMedDate not supplied

Analysis of the collagen VI assemblies associated with Sorsby's fundus dystrophy.

Age-related macular degeneration is the leading cause of blindness in the Western world, and the pathophysiology of the condition is largely unknown. However, it shares many clinical and pathological features with Sorsby's fundus dystrophy (SFD), an autosomal dominant disease, known to be associated with mutations in the TIMP-3 gene. In Bruch's membrane of both conditions, there are molecular assemblies with distinct transverse bands occurring with a periodicity of about 100 nm. Similar assemblies were also found in the vitreous of a patient with full-thickness macular holes and were identified as being made of collagen VI. The assemblies found in the eye with SFD can be classified into two types, both with a 105-nm axial repeat, but one showing pairs of narrow bands about 30 nm apart and the other showing a single broad band in every repeat. By comparison with the assemblies in the vitreous, collagen VI is considered to be the most likely protein in these assemblies. Furthermore, both of the assemblies associated with SFD can be explained in terms of collagen VI tetramers, one in which the tetramers bind to the mutant tissue inhibitor of metalloproteinases-3 (the gene product of TIMP-3) and the other in which little or no binding occurs. TIMP-3 bound to collagen VI may be more resistant to degradation and create an imbalance between the normal amount of TIMP-3 and matrix metalloproteinases (the substrate of TIMPs) in Bruch's membrane with consequent disruption of the normal metabolic processes. Understanding the structure of these collagen VI/TIMP assemblies in Bruch's membrane may prove to be important for understanding the pathophysiology of age-related macular degeneration.

Aged↗

PubMed2000

Assembly of complex organelles: pilus biogenesis in gram-negative bacteria as a model system.

Pathogenic bacteria assemble a variety of adhesive structures on their surface for attachment to host cells. Some of these structures are quite complex. For example, the hair-like organelles known as pili or fimbriae are generally composed of several components and often exhibit composite morphologies. In gram-negative bacteria assembly of pili requires that the subunits cross the cytoplasmic membrane, fold correctly in the periplasm, target to the outer membrane, assemble into an ordered structure, and cross the outer membrane to the cell surface. Thus, pilus biogenesis provides a model for a number of basic biological problems including protein folding, trafficking, secretion, and the ordered assembly of proteins into complex structures. P pilus biogenesis represents one of the best-understood pilus systems. P pili are produced by 80-90% of all pyelonephritic Escherichia coli and are a major virulence determinant for urinary tract infections. Two specialized assembly factors known as the periplasmic chaperone and outer membrane usher are required for P pilus assembly. A chaperone/usher pathway is now known to be required for the biogenesis of more than 30 different adhesive structures in diverse gram-negative pathogenic bacteria. Elucidation of the chaperone/usher pathway was brought about through a powerful combination of molecular, biochemical, and biophysical techniques. This review discusses these approaches as they relate to pilus assembly, with an emphasis on newer techniques.

Fimbriae, Bacterial↗

PubMedDate not supplied

Cell-assembly coding in several memory processes.

The present paper discusses why the cell assembly, i.e., an ensemble population of neurons with flexible functional connections, is a tenable view of the basic code for information processes in the brain. The main properties indicating the reality of cell-assembly coding are neurons overlaps among different assemblies and connection dynamics within and among the assemblies. The former can be detected as multiple functions of individual neurons in processing different kinds of information. Individual neurons appear to be involved in multiple information processes. The latter can be detected as changes of functional synaptic connections in processing different kinds of information. Correlations of activity among some of the recorded neurons appear to change in multiple information processes. Recent experiments have compared several different memory processes (tasks) and detected these two main properties, indicating cell-assembly coding of memory in the working brain. The first experiment compared different types of processing of identical stimuli, i.e., working memory and reference memory of auditory stimuli. The second experiment compared identical processes of different types of stimuli, i.e., discriminations of simple auditory, simple visual, and configural auditory-visual stimuli. The third experiment compared identical processes of different types of stimuli with or without temporal processing of stimuli, i.e., discriminations of elemental auditory, configural auditory-visual, and sequential auditory-visual stimuli. Some possible features of the cell-assembly coding, especially "dual coding" by individual neurons and cell assemblies, are discussed for future experimental approaches.

Animals↗

PubMed1995

In vitro assembly of cowpea chlorotic mottle virus from coat protein expressed in Escherichia coli and in vitro-transcribed viral cDNA.

The small spherical plant virus, cowpea chlorotic mottle virus (CCMV), provides an ideal system to examine spherical virus assembly. We have modified the CCMV in vitro assembly system to produce virions from coat protein expressed in Escherichia coli and viral RNA transcribed in vitro from full-length cDNAs. Examination of the in vitro-assembled particles with cryoelectron microscopy and image reconstruction techniques demonstrates that the particles are indistinguishable from plant purified particles at 2.5 nm resolution. Mutational analysis of the coat protein N- and C-terminal extensions demonstrate their respective roles in virus assembly. The N-terminus is required for assembly of RNA containing particles but not for the assembly of empty virions. The C-terminus is essential for coat protein dimer formation and particle assembly.

Base Sequence↗

PubMed1992

Synthesis and assembly of soybean beta-conglycinin in vitro.

The construction of SP6-derived expression plasmids that encode normal and modified beta-conglycinin subunits is described. With the exception of an additional methionine at their NH2-terminal ends and the lack of glycans, the normal subunits synthesized at the direction of these plasmids corresponded to mature alpha and beta subunits isolated from soybean seeds. The subunits assembled into trimers in vitro that were equivalent in size to those formed in vivo. This result shows that the glycans are not required either for protein folding or oligomer assembly. Subunits produced from other plasmids, which had modifications in a highly conserved hydrophobic region in the COOH-terminal end of the subunits, either did not assemble or assembled at an extremely low rate compared to unmodified subunits. Structural changes at the more hydrophilic NH2-terminal end had mixed effects. Several subunits modified in this region assembled into trimers at rates that were either equal or greater than those for normal alpha subunits. Others assembled less completely than the normal subunits. Our results indicate that the in vitro synthesis and assembly assay will be useful in evaluating structure-function relationships in modified beta-conglycinin subunits. The results also show that structural changes at the NH2-terminal end of the subunits are tolerated to a greater extent than modifications in the hydrophobic conserved region in the COOH-terminal half of the subunits, and this information will be useful in efforts to improve soybean quality.

Amino Acid Sequence↗

PubMed1995

The in vitro assembly of the NADPH-protochlorophyllide oxidoreductase in pea chloroplasts.

The NADPH-protochlorophyllide oxidoreductase (pchlide reductase, EC 1.6.99.1) is the major protein in the prolamellar bodies (PLBs) of etioplasts, where it catalyzes the light-dependent reduction of protochlorophyllide to chlorophyllide during chlorophyll synthesis in higher plants. The suborganellar location in chloroplasts of light-grown plants is less clear. In vitro assays were performed to characterize the assembly process of the pchlide reductase protein in pea chloroplasts. Import reactions employing radiolabelled precursor protein of the pchlide reductase showed that the protein was efficiently imported into fully matured green chloroplasts of pea. Fractionation assays following an import reaction revealed that imported protein was targeted to the thylakoid membranes. No radiolabelled protein could be detected in the stromal or envelope compartments upon import. Assembly reactions performed in chloroplast lysates showed that maximum amount of radiolabelled protein was associated to the thylakoid membranes in a thermolysin-resistant conformation when the assays were performed in the presence of hydrolyzable ATP and NADPH, but not in the presence of NADH. Furthermore, membrane assembly was optimal at pH 7.5 and at 25 degrees C. However, further treatment of the thylakoids with NaOH after an assembly reaction removed most of the membrane-associated protein. Assembly assays performed with the mature form of the pchlide reductase, lacking the transit peptide, showed that the pre-sequence was not required for membrane assembly. These results indicate that the pchlide reductase is a peripheral protein located on the stromal side of the membrane, and that both the precursor and the mature form of the protein can act as substrates for membrane assembly.

Adenosine Triphosphate↗

PubMed1990

Microtubule assembly and oscillations induced by flash photolysis of caged-GTP.

Microtubule assembly and oscillations have been induced using the rapid liberation of GTP by UV flash photolysis of caged-GTP and monitored by time-resolved X-ray scattering. The flash photolysis method of achieving assembly conditions is much faster than the temperature jump method used earlier (msec vs. s range). However, the structural transitions and their rates are similar to those described previously. This means that the rates of the transitions in microtubule assembly observed before are determined by the protein itself, and not by the rate at which assembly conditions are induced. The advantages and limitations of using the photolysis of caged-GTP in microtubule assembly studies are compared with temperature jump methods. Caged-GTP itself reduces the rate of microtubule assembly and oscillations at mM concentrations, consistent with a weak interaction between the nucleotide analogue and the protein. X-rays are capable of slowly liberating GTP and other breakdown products from caged-GTP, even in the absence of UV flash photolysis, thus causing an apparent "X-ray-induced" microtubule assembly. This effect depends on the X-ray dose but is independent of the caged-GTP concentrations used here (mM range), suggesting that the breakdown of caged-GTP is caused not by the direct absorption of X-rays by the compound but by another intermediate reaction such as the generation of radicals by the X-rays.

Animals↗

PubMed1986

Phorbol ester induces rapid actin assembly in neutrophil leucocytes independently of changes in [Ca2+]i and pHi.

The phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), at nanomolar concentrations, induces rapid (t1/2 approximately 30 s) protrusion of multiple petal-shaped lamellae by neutrophil leucocytes. Lamellae are richly endowed with actin filaments as determined by the localization of rhodamine-phalloidin, suggesting extensive assembly at the cell cortex. Direct measurement of the proportion of total cell actin which is polymerized, by using a deoxyribonuclease I inhibition assay, indicates that the proportion of polymerized actin approximately doubles, and that assembly initiated by 30 nM TPA occurs with no obvious lag phase and with a t1/2 of about 30 s. A half-maximal response was induced at 2 nM TPA. Since both actin assembly and protrusion of lamellae are completely inhibited by 10(-6) M cytochalasin D, protrusion of lamellae is presumably dependent on actin filament assembly. To examine whether TPA induces actin assembly via changes in [Ca2+]i or pHi, these parameters were monitored in cells loaded with the fluorescent indicators quin2 and quene1 respectively. Addition of TPA caused no change in [Ca2+]i but a biphasic change in pHi. To examine further the potential role of ionic changes in regulation of actin assembly, the morphological responses of cells to TPA were monitored in severely Ca2+-depleted cells, or cells in which pHi had been experimentally raised or lowered by simultaneous additions of a weak base (NH4Cl) or weak acid (CH3COONa) respectively. The protrusion of lamellae induced by TPA was completely unaffected by these experimental manipulations indicating that TPA can directly regulate actin assembly, and hence morphology, by mechanisms essentially independent of [Ca2+]i and pHi.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

PubMed2006

Predicting understorey structure from the presence and composition of canopies: an assembly rule for marine algae.

Assembly rules provide a useful framework for predicting patterns of community assembly under defined environmental conditions. Habitat created by canopy-forming algae (such as kelps) provides a promising system for identifying assembly rules because canopies typically have a large and predictable influence on understorey communities. Across >1,000 km of subtidal South Australian coastline, we identified natural associations between assemblages of understorey algae and (1) monospecific canopies of Ecklonia radiata, (2) canopies comprised of E. radiata mixed with Fucales (Cystophora spp. and Sargassum spp.), and (3) gaps among canopies of algae. We were able to recreate these associations with experimental tests that quantified the assembly of understorey algae among these three habitat types. We propose the assembly rule that understorey communities on subtidal rocky coast in South Australia will be (1) monopolised by encrusting coralline algae beneath monospecific canopies of E. radiata, (2) comprised of encrusting corallines, encrusting non-corallines, and sparse covers of articulated corallines, beneath mixed E. radiata-Fucales canopies, and (3) comprised of extensive covers of articulated corallines and filamentous turfs, as well as sparse covers of foliose algae and juvenile canopy-formers, within gaps. Consistencies between natural patterns and experimental effects demonstrate how algal canopies can act as a filter to limit the subsets of species from the locally available pool that are able to assemble beneath them. Moreover, the subsets of species that assemble to subtidal rocky substrata in South Australia appear to be predictable, given knowledge of the presence and composition of canopies incorporating E. radiata.

Analysis of Variance↗

PubMed1989

Alkaline pH favors microtubule self-assembly in surf clam, Spisula solidissima, oocyte extracts.

Microtubule assembly in surf clam oocytes is dependent upon events that occur during fertilization. Prior to fertilization there are few, if any microtubules, but within minutes after fertilization microtubules assemble to form the meiotic apparatus. This study demonstrates that the assembly of microtubules after fertilization may be dependent on the fertilization-induced pH change of the cytoplasm. Since the magnitude of the intracellular pH (pHi) change in Spisula oocytes has not been determined, surf clam microtubule assembly was examined at pH values that reflect the pHi change that occurs during sea urchin fertilization. The results indicate that microtubule assembly in crude oocyte extracts is favored at alkaline pH. In contrast, purified surf clam tubulin assembles to a greater extent at pH 6.6 than at pH 7.2. These results reveal that the tubulin in unfertilized oocytes can assemble into microtubules at pH 6.6 but that they are prevented from doing so by pH-dependent cytoplasmic regulatory factors in the oocyte.

Animals↗

PubMed1990

Localization of anti-clathrin antibody in the sarcomere and sensitivity of myofibril structure to chloroquine suggest a role for clathrin in myofibril assembly.

Immunofluorescence microscopy has been used to demonstrate that X22, a monoclonal antibody specific for clathrin heavy chain, localizes in repetitive bands that appear soon after the fusion of skeletal myoblasts into multinucleate fibers. This organization has been found in cultures containing myotubes that develop in vitro from explants of newborn rat hindlimb cells and in myotubes derived from the L8E63 myogenic line. Bands were also prominent in skinned fibers prepared from adult rat soleus muscle and in cardiac myocytes grown in vitro from 4-day heart ventricles. Immunofluorescence banding was localized in the sarcomere as a doublet, with one element on either side of the Z line. Evidence that supports the conclusion that the reaction with X22 antibody is specific and indicative of the localization of clathrin in the sarcomere includes: (1) Identical titration of X22 antibody reactivity with the determinant in coated vesicles and in the sarcomere. (2) Conditions (eg., pH and Tris) that disrupt clathrin baskets or prevent its assembly likewise disrupt the localization of X22 in bands. (3) Chloroquine inhibits both the normal trafficking of clathrin in the cell and X22 banding in the sarcomere. (4) Immunoblot analysis of myotube lysates reveals a single band with an electrophoretic mobility identical to the 180,000-Da clathrin heavy chain. (5) The assembly of clathrin into sarcomeric bands occurs early in the development of the myofibrillar apparatus. Quantitation of the appearance of X22 banding in primary cultures of myotubes indicates that it precedes that of other myofibrillar proteins and that assembly takes place in the following order: X22, titin, myosin heavy chain, actin, and desmin. The assembly of myosin, titin, and actin into sarcomeric bands, as well as X22, is inhibited by chloroquine. Upon prolonged exposure to chloroquine previously assembled proteins are drastically reduced or no longer evident in the sarcomere. On the basis of these results and considering the role of clathrin in intracellular transport and its capacity to interact with actin and alpha-actinin, we suggest that clathrin may have diverse roles in the assembly, integrity, and functioning of the sarcomere and its integration with the sarcolemma. The early organization of X22 into bands further suggests that clathrin may also function early in the assembly of the contractile system.

Actins↗

PubMed1985

Assembly of vimentin in vitro and its implications concerning the structure of intermediate filaments.

After dialysis against 10 mM-Tris-acetate (pH 8.5), vimentin that has been purified in the presence of urea is present in the form of tetrameric 2 to 3 nm X 48 nm rods known as protofilaments. These building blocks in turn polymerize into intermediate filaments (10 to 12 nm diameter) when they are dialyzed against a solution of physiological ionic strength and pH. By varying the ionic conditions under which polymerization takes place, we have identified two classes of assembly intermediates whose structures provide clues as to how an intermediate filament may be constructed. The structure of the first class, seen when assembly takes place at 10 to 20 mM-salt at pH 8.5, strongly suggests that one of the initial steps of filament assembly is the association of protofilaments into pairs with a half-unit axial stagger. Increasing the ionic strength of the assembly buffer leads to the emergence of short, full-width intermediate filaments at approximately 50 mM-salt at pH 8.5. In the presence of additional protofilaments, these short filaments elongate to many micrometers when the ionic strength and pH are further adjusted to physiological levels. The electron microscope images of the assembly intermediates suggest that vimentin-containing intermediate filaments are made up of eight protofilaments, assembled such that there is an approximately 22 nm axial stagger between neighboring protofilaments. We propose that this half-unit staggering of protofilaments is a fundamental feature of intermediate filament structure and assembly, and that it could account for the 20 to 22 nm axial repeat seen in all intermediate filaments examined so far.

Animals↗

PubMed1988

The tobacco mosaic virus assembly origin RNA. Functional characteristics defined by directed mutagenesis.

The in vitro reassembly of tobacco mosaic virus (TMV) begins with the specific recognition by the viral coat protein disk aggregate of an internal TMV RNA sequence, known as the assembly origin (Oa). This RNA sequence contains a putative stem-loop structure (loop 1), believed to be the target for disk binding in assembly initiation, which has the characteristic sequence AAGAAGUCG exposed as a single strand at its apex. We show that a 75-base RNA sequence encompassing loop 1 is sufficient to direct the encapsidation by TMV coat protein disks of a heterologous RNA fragment. This RNA sequence and structure, which is sufficient to elicit TMV assembly in vitro, was explored by site-directed mutagenesis. Structure analysis of the RNA identified mutations that appear to effect assembly via a perturbation in RNA structure, rather than by a direct effect on coat protein binding. The binding of the loop 1 apex RNA sequence to coat protein disks was shown to be due primarily to its regularly repeated G residues. Sequences such as (UUG)3 and (GUG)3 are equally effective at initiating assembly, indicating that the other bases are less functionally constrained. However, substitution of the sequences (CCG)3, (CUG)3 or (UCG)3 reduced the assembly initiation rate, indicating that C residues are unfavourable for assembly. Two additional RNA sequences within the 75-base Oa sequence, both of the form (NNG)3, may play subsidiary roles in disk binding. RNA structure plays an important part in permitting selective protein-RNA recognition, since altering the RNA folding close to the apex of the loop 1 stem reduces the rate of disk binding, as does shortening the stem itself. Whereas the RNA sequence making up the hairpin does not in general affect the specificity of the protein-RNA interaction, it is required to present the apex signal sequence in a special conformation. Mechanisms for this are discussed.

Base Sequence↗

PubMed1990

Novel second-site suppression of a cold-sensitive defect in phage P22 procapsid assembly.

The DNA packaging portal of the phage P22 procapsid is formed of 12 molecules of the 90,000 dalton gene 1 protein. The assembly of this dodecameric complex at a unique capsid vertex requires scaffolding subunits. The mechanism that ensures the location of the 12-fold symmetrical portal at only one of the 12 5-fold vertices of an icosahedral virus capsid presents a unique assembly problem, which, in some viruses, is solved by the portal also acting as initiator of procapsid assembly. Phage P22 procapsids, however, are formed in the absence of the portal protein. The 1-csH137 mutation prevents the incorporation of the portal protein into procapsids. In a mixed infection with cs+ phage, the mutant subunits are able to form functional portals, suggesting that the cold-sensitivity does not affect portal-portal interactions, but affects the interaction of portal subunits with some other molecular species involved in the initiation of portal assembly. Interestingly, the cs defect is suppressed by temperature-sensitive folding mutations at four sites in the P22 tailspike gene 9. The suppression is allele-specific; other tailspike tsf mutations fail to suppress the cs defect. Translation through a suppressor site is required for suppression. This observation is unexpected, since analysis of nonsense mutations in this gene indicates that it is not required for procapsid assembly. Examination of the nucleic acid sequences in the neighborhood of each of the suppressor sites shows significant sequence similarity with the scaffolding gene translational initiation region on the late message. This supports a previously proposed model, in which procapsid assembly is normally initiated in a region on the late messenger RNA that includes the gene 8 start site. By this model, the suppressor mutations may be acting through protein-RNA interactions, changing sequences that identify alternative or competing sites at which the mutant portal subunits may be organized for assembly into the differentiated vertex of the phage capsid.

Alleles↗