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1.
Remarkable progress has been made during the past ten years in elucidating the structure of the bacteriophage T4 tail by a combination of three-dimensional image reconstruction from electron micrographs and X-ray crystallography of the components. Partial and complete structures of nine out of twenty tail structural proteins have been determined by X-ray crystallography and have been fitted into the 3D-reconstituted structure of the "extended" tail. The 3D structure of the "contracted" tail was also determined and interpreted in terms of component proteins. Given the pseudo-atomic tail structures both before and after contraction, it is now possible to understand the gross conformational change of the baseplate in terms of the change in the relative positions of the subunit proteins. These studies have explained how the conformational change of the baseplate and contraction of the tail are related to the tail's host cell recognition and membrane penetration function. On the other hand, the baseplate assembly process has been recently reexamined in detail in a precise system involving recombinant proteins (unlike the earlier studies with phage mutants). These experiments showed that the sequential association of the subunits of the baseplate wedge is based on the induced-fit upon association of each subunit. It was also found that, upon association of gp53 (gene product 53), the penultimate subunit of the wedge, six of the wedge intermediates spontaneously associate to form a baseplate-like structure in the absence of the central hub. Structure determination of the rest of the subunits and intermediate complexes and the assembly of the hub still require further study.  相似文献   

2.
The collar and whiskers of bacteriophage T4 extend outward from the top of the tail and play a role in regulating retraction of the tail fibers (Conley &; Wood, 1975). The collar and whiskers also are required for efficient tail fiber attachment during phage assembly. The structural gene for the collar/whisker protein is called wac. In vitro, infected-cell extracts that contain tail fibers activate whiskerless (wac) tail fiberless particles and ordinary (wac+) tail fiberless particles at equal rates if the extracts contain the wac+ gene product. However, extracts that contain tail fibers but no wac+ gene product activate wac particles about ten times more slowly. In vivo, whiskers are not essential for plaque formation, but a wac mutation causes a delay in the appearance of intracellular phage and a fivefold decrease in the burst size of infectious particles.The effect of the whiskers on tail fiber attachment is due to an interaction between the whisker and the distal half of the tail fiber, similar if not identical to the interaction that controls tail fiber retraction in complete phage. The following observations support this view: a slow rate of in vitro tail fiber attachment similar to that described above is seen with wac+ particles when they are pretreated with anti-whisker serum, or when the tail fibers carry a mutational alteration in gp36, a structural protein in the distal half fiber near the central kink. Lack of whiskers does not affect the slow rate of attachment of proximal half fibers to the baseplate of fiberless particles, but lack of whiskers greatly decreases the rate at which particles with attached proximal half fibers are activated by addition of distal half fibers. Since whiskers normally are attached to the phage only after head—tail union (Coombs &; Eiserling, 1977; Terzaghi et al., 1978), these findings explain why tail fibers do not attach efficiently to the baseplates of free tails.  相似文献   

3.
The T4D bacteriophage gene 28 product is a component of the central plug of the tail baseplate, as shown by the following two independent lines of evidence. (i) A highly sensitive method for radioactive labeling of only tail baseplate plug components was developed. These labeled plug components were incorporated by a complementation procedure into new phage particles and were analyzed by radioautography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three new structural proteins were found in addition to the three known tail plug proteins (i.e., gP29, gP27, and gP5). One of the three newly identified components had a molecular weight of 24,000 to 25,000 and appeared to be a product of T4D gene 28. (ii) Characterization of mutants of Escherichia coli bacteriophage T4D which produced altered gene 28 products also indicated that the gene 28 product was a viral tail component. T4D 28ts phage particles produced at the permissive temperature had altered heat labilities compared with parent T4D particles. We isolated a single-step temperature revertant of T4D 28ts and found that it produced phage particles which phenotypically resembled the original T4D particles. Since the properties of the phage baseplate components usually determine heat lability, these two changes in physical stability after two sequential single mutations in gene 28 supported the other evidence that the gene 28 product was a viral baseplate component. Also, compared with parent T4D particles, T4D 28ts and T4D 28am viral particles adsorbed at different rates to various types of host cells. In addition, T4D 28ts particles exhibited a different host range than parent T4D particles. This T4D mutant formed plaques with an extremely low efficiency on all E. coli K-12 strains tested. We found that although T4D 28ts particles adsorbed rapidly and irreversibly to the E. coli K-12 strains, as judged by gene rescue experiments, these particles were not able to inject their DNA into the E. coli K-12 strains. On the other hand, the T4D 28ts revertant had a plating efficiency on E. coli K-12 strains that was quite similar to the plating efficiency of the original parent, T4D. These properties of phage particles containing an altered gene 28 product supported the analytical finding that the gene 28 product is a structural component of the central plug of the T4D tail baseplate. They also indicated that this component plays a role in both host cell recognition and viral DNA injection.  相似文献   

4.
Normal particles of bacteriophage T4 have six long tail fibers attached to a hexagonal baseplate. T4 particles having various complements of tail fibers were prepared by in vitro addition of fibers to fiberless particles, and the infectivity of the particles was determined. Particles having fewer than six fibers (partially fibered) were found to have a decreased probability of infection. Partially fibered particles having T4 fibers were completed by addition of T6 fibers, and the infectivity was determined on a host that lacked the T6 tail fiber receptor. Attachment of the additional fibers increased the infectivity even though the T6 fibers could not bind to the host cell. The infectivity of particles having mixtures of T4 and T6 fibers was determined on cells having only one type of receptor. The results indicated that particles bound by only three fibers have a low probability of infection. The effect of thermolabile baseplate mutations was also examined. Studies of partially fibered particles and particles with mixtures of fibers indicated that particles with altered baseplates have a less stringent requirement for binding of the tail fibers for infection.  相似文献   

5.
In wild type bacteriophage T4 the long tail fibres serve both in the initial attachment of the phage to its host and in the triggering of tail contraction. A two-step model for the control of triggering suggests that particles lacking the product of gene 9, which are also structurally fibreless, might be infective. This is shown to be the case, even though such phage do not plate on restrictive strains of bacteria. However, starting from phage carrying an amber mutation in gene 9 it is easy to isolate additional mutations which, under restrictive conditions, permit fibreless plating (pfp mutations). Three such pfp mutations, having also a temperature-sensitive phenotype, have been isolated and shown to map in genes coding for structural components of the baseplate. The mode of action of these pfp mutations is not clear, though they certainly destabilize the baseplate, thereby making triggering easier. The pfp mutations are effective only when in combination with an amber mutation in gene 9 and not with amber mutations in tail fibre genes, establishing the essentially inhibitory nature of the control of triggering exercised by gene 9 product.  相似文献   

6.
The tail lysozyme complex of bacteriophage T4   总被引:1,自引:0,他引:1  
The tail baseplate of bacteriophage T4 contains a structurally essential, three-domain protein encoded by gene 5 in which the middle domain possesses lysozyme activity. The gene 5 product (gp5) undergoes post-translational cleavage, allowing the resultant N-terminal domain (gp5*) to assemble into the baseplate as a trimer. The lysozyme activity of the undissociated cleaved gp5 is inhibited until infection has been initiated, when the C-terminal portion of the molecule is detached and the rest of the molecule dissociates into monomers. The 3D structure of the undissociated cleaved gp5, complexed with gp27 (another component of the baseplate), shows that it is a cell-puncturing device that functions to penetrate the outer cell membrane and to locally dissolve the periplasmic cell wall.  相似文献   

7.
Identification of P48 and P54 as components of bacteriophage T4 baseplates.   总被引:13,自引:8,他引:5  
The involvement of two bacteriophage T4 gene products in the initiation of T4 tail tube and sheath polymerization on mature baseplates has been studied by radioautography of acrylamide gels of various partially completed tail structures. The products of genes 48 and 54 (P48[the nomenclature P48 refers to the protein product of bacteriophage T4 gene 48] and P54), which are known to be required for the synthesis of mature baseplates, have been shown to be structural components of the baseplate. These gene products have molecular weights of 42,000 and 33,000, respectively. The addition of P54 to the baseplate not only permits the polymerization of the core protein, P19, onto the baseplate, but also caused the disappearance of a polypeptide of molecular weight about 15,000 from the supernatant fraction of infected cells. Another gene product, P27, has been identified in the crude extracts of infected cells. This gene product, which is required for the synthesis of baseplate structures, has the same mobility as one of the unidentified structural polypeptides of the baseplate and is therefore probably also a baseplate component.  相似文献   

8.
Summary Formation of both the tail fiber and the baseplate of bacteriophage T4 depends on the product of T4 gene 57. A single amber mutation in that gene causes loss of two T4-specific proteins. Their molecular weights are 18,000 and about 6,000, respectively, based on their electrophoretic mobilities in SDS-polyacrylamide gels. E. coli carrying a cloned T4 DNA fragment of about 700 basepairs, which directs the synthesis of the smaller protein only, specifically supports the growth of gene 57 amber mutants. We conclude that the small protein is a functional product of gene 57.Abbreviations Am ampicillin - Cm chloramphenicol - Tet tetracycline - SDS sodium dodecyl sulfate - bp basepairs - wt wildtype - Su suppressor - Km kanamycin - ds double stranded - ss single stranded - SDS-PAGE SDS-polyacrylamide gel electrophoresis  相似文献   

9.
Protein interactions in the assembly of the baseplate have been investigated. The baseplate of the phage T4 tail consists of a hub and six wedges which surround the former. Both reversible and irreversible interactions were found. Reversible association includes gp5 and gp27 (gp: gene product) which form a complex in a pH-dependent manner and gp18 polymerization, i.e. the tail sheath formation depends on the ionic strength. These reversible interactions were followed by irreversible or tight binding which pulls the whole association reaction to complete the assembly. The wedge assembly is strictly ordered which means that if one of the seven wedge proteins is missing, the assembly proceeds to that point and the remaining molecules stay non-associated. The strictly sequential assembly pathway is suggested to be materialized by successive conformational change upon binding, which can be shown by proteolytic probe.  相似文献   

10.
The tail of bacteriophage T4 undergoes large structural changes upon infection while delivering the phage genome into the host cell. The baseplate is located at the distal end of the contractile tail and plays a central role in transmitting the signal to the tail sheath that the tailfibers have been adsorbed by a host bacterium. This then triggers the sheath contraction. In order to understand the mechanism of assembly and conformational changes of the baseplate upon infection, we have determined the structure of an in vitro assembled baseplate through the three-dimensional reconstruction of cryo-electron microscopy images to a resolution of 3.8 Å from electron micrographs. The atomic structure was fitted to the baseplate structure before and after sheath contraction in order to elucidate the conformational changes that occur after bacteriophage T4 has attached itself to a cell surface. The structure was also used to investigate the protease digestion of the assembly intermediates and the mutation sites of the tail genes, resulting in a number of phenotypes.  相似文献   

11.
Summary The proteins synthesized in Escherichia coli B cells after infection with various T4 bacteriophage tail baseplate mutants were analysed by the immunoblotting method for the presence of the 15 Kilodalton lysozyme found in phage T4 particles. Using three different antisera: anti-phage, anti-baseplate and anti-15K lysozyme, it has been found that the 15K lysozyme is not present in lysates of bacteria infected with T4 gene 25 amber mutants. The 15K lysozyme was also found to be expressed in E. coli B cells transformed with a plasmid containing only a small portion of the T4 genome but which included T4 gene 25. These observations indicate that the 15K lysozyme is the gene 25 product.  相似文献   

12.
BACKGROUND: The T4 bacteriophage consists of a head, filled with double-stranded DNA, and a complex contractile tail required for the ejection of the viral genome into the Escherichia coli host. The tail has a baseplate to wh?ch are attached six long and six short tail fibers. These fibers are the sensing devices for recognizing the host. When activated by attachment to cell receptors, the fibers cause a conformational transition in the baseplate and subsequently in the tail sheath, which initiates DNA ejection. The baseplate is a multisubunit complex of proteins encoded by 15 genes. Gene product 9 (gp9) is the protein that connects the long tail fibers to the baseplate and triggers the tail contraction after virus attachment to a host cell. RESULTS: The crystal structure of recombinant gp9, determined to 2.3 A resolution, shows that the protein of 288 amino acid residues assembles as a homotrimer. The monomer consists of three domains: the N-terminal domain generates a triple coiled coil; the middle domain is a mixed, seven-stranded beta sandwich with a topology not previously observed; and the C-terminal domain is an eight-stranded, antiparallel beta sandwich having some resemblance to 'jelly-roll' viral capsid protein structures. CONCLUSIONS: The biologically active form of gp9 is a trimer. The protein contains flexible interdomain hinges, which are presumably required to facilitate signal transmission between the long tail fibers and the baseplate. Structural and genetic analyses show that the C-terminal domain is bound to the baseplate, and the N-terminal coiled-coil domain is associated with the long tail fibers.  相似文献   

13.
The length of the T4 tail is precisely regulated in vivo at the time of polymerization of the tail core protein onto the baseplate. Since no mutations which alter tail length have been identified, a study of in vivo-assembled tail cores was begun to determine whether the structural properties of assembled cores would reveal the mechanism of length regulation. An assembly intermediate consisting of a core attached to a baseplate (core-baseplate) was purified from cells infected with a T4 mutant in gene 15. When core-base plates were treated with guanidine hydrochloride, cores were released from baseplates. The released cores had the same mean length as cores attached to baseplates. Electron micrographs of these cores showed partial penetration of negative stain into one end, and, at the opposite end, a modified tip which often appeared as a short fiber projecting from the core. When cores were purified and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two minor proteins and the major core protein were detected. One minor protein, the product of gene 48 (gp48), was present in at least 72% of the amount found in core-baseplates, relative to the amount of the major core protein. These findings suggest that cores contain a fibrous structure, possibly composed of gp48, which may form a "ruler" that specifies the length of the T4 tail.  相似文献   

14.
Purified preparations of complete T4 bacteriophage, tail fiberless particles, whole tail fibers and four tail fiber precursors were dissociated by heating briefly at 100 °C in 1% sodium dodecyl sulfate containing 1% mercaptoethanol. Analysis of the dissociated structures by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol revealed two high molecular weight (150,000 and 123,000 daltons) polypeptides as major tail fiber components. These two components could be easily identified by autoradiography of sodium dodecyl sulfate gels of radioactively labeled infected cell extracts. The larger of the two was missing from extracts of cells infected with gene 34 amber mutants, and the smaller from extracts of cells infected with gene 37 amber mutants. It is concluded that the two components represent the products of genes 34 and 37 (P34 and P37), respectively. Molecular weight calculations indicate that two copies of each polypeptide are present in each complete tail fiber. Amber mutations in genes 38 and 57 were found to affect the apparent solubility of P34 and P37 and their resistance to dissociation in cold sodium dodecyl sulfate, but not their synthesis. Based on these results, the previously reported pathway of tail fiber assembly (King & Wood, 1969) has been reformulated in more detail.  相似文献   

15.
Products of two bacteriophage T4D genes, 26 and 51, both known to be essential for the formation of the central hub of the phage tail baseplate, have been partially characterized chemically, and their biological role has been examined. The gene 26 product was found to be a protein with a molecular size of 41,000 daltons and the gene 51 product a protein of 16,500 daltons. The earlier proposal (L. M. Kozloff and J. Zorzopulos, J. Virol. 40:635-644), from observations of a 40,000-dalton protein in labeled hubs, that the gene 26 product is a structural component of the baseplate, has been confirmed. The gene 51 product, not yet detected in phage particles, appears from indirect evidence also to be a structural component of the baseplate hub. These current conclusions about the gene 26 and 51 products are based on properties of T4 mutant particles containing altered gene 26 or 51 products and include (i) changes in heat lability, (ii) changes in adsorption rates, and (iii) changes in plating efficiencies on different hosts, and with the results of previous isotope incorporation experiments indicate that T4 particles contain three copies of the gene 26 product and possibly one or at most two copies of the gene 51 product. Properties of these mutant particles indicate that the gene 26 product, together with the other hub components such as the gene 28 product, plays a critical role in phage DNA injection into the host cell, whereas the 51 product seems essential in initiating baseplate hub assembly.  相似文献   

16.
The baseplate of bacteriophage T4 is a complex structure containing at least 14 different structural proteins. It undergoes a transition from a hexagonal to a star-shaped configuration during infection of the host bacterial cell. We have used a combination of genetics and image processing of electron micrographs to analyse both the wild-type structure and a series of mutant structures lacking specific gene products. Besides describing the basic anatomy of the hexagon and star configurations, we have been able to locate the products of genes 9, 11 and 12.Gene 9 product occupies a peripheral position in hexagons and stars consistent with its providing a binding site for the long tail fibres. Gene 11 product in the hexagon forms the distal part of the tail pin, which folds out to form the point of the hexagram in the star configuration. Gene 12 product is visualized as an extended 350 Å fibre in stars and broken baseplates but appears to have a more compact configuration in hexagons and intact phage.We demonstrate the structural relationship between the hexagonal and starshaped configurations and show how the positions of the specific gene products alter as a result of the structural transition. We suggest a speculative model for the role of gene 9 and gene 12 products in triggering the rearrangement of the baseplate and tail contraction.  相似文献   

17.
L M Kozloff  M Lute    L K Crosby 《Journal of virology》1975,16(6):1391-1400
Two different proteins with high affinities for the pteridine ring of folic acid have been used to determine the location of this portion of the folate molecule in the tail plate of T4D and other T-even bacteriophage particles. The two proteins used were (i) antibody specific for folic acid and (ii) the folate-binding protein from bovine milk. Both proteins were examined for their effect on various intact and incomplete phage particles. Intact T2H was weakly inactivated by the antiserum but not by the milk protein. No other intact T-even phage, including T4D, was affected by these two proteins. When incomplete T4D particles were exposed in an in vitro morphogenesis system, it was found that neither of the two proteins affected either the addition of the long tail fibers to fiberless particles or the addition of tail cores to tail plates. On the other hand, these two proteins specifically blocked the addition of T4D gene 11 product to the bottom of T4D baseplates. After the addition of the gene 11 protein, these two reagents did not inhibit the further addition of the gene 12 protein to the baseplate. It can be concluded that the phage folic acid is a tightly bound baseplate constituent and that the pteridine portion of the folic acid is largely covered by the gene 11 protein.  相似文献   

18.
The protein products of at least 21 phage genes are needed for the formation of the tail of bacteriophage T4. Cells infected with amber mutants defective in these genes are blocked in the assembly process. By characterizing the intermediate structures and unassembled proteins accumulating in mutant-infected cells, we have been able to delineate most of the gene-controlled steps in tail assembly. Both the organized structures and unassembled proteins serve as precursors for in vitro tail assembly. We review here studies on the initiation, polymerization, and termination of the tail tube and contractile sheath and the genetic control of these processes. These studies make clear the importance of the baseplate; if baseplate formation is blocked (by mutation) the tube and sheath subunits remain essentially unaggregated, in the form of soluble subunits. Seventeen of the 21 tail genes specify proteins involved in baseplate assembly. The genes map contiguously in two separate clusters, one of nine genes and the other of eight genes. Recent studies show that the hexagonal baseplate is the end-product of two independent subassembly pathways. The proteins of the first gene cluster interact to form a structure which probably represents one-sixth of the outer radius. The products of the other gene cluster interact to form the central part of the baseplate. Most of the phage tail precursor proteins appear to be synthesized in a non-aggregating form; they are converted to a reactive form upon incorporation into preformed substrate complexes, without proteolytic cleavage. Thus reactive sites are limited to growing structures.  相似文献   

19.
Chemically Induced Cofactor Requirement for Bacteriophage T4D   总被引:1,自引:0,他引:1       下载免费PDF全文
The treatment of bacteriophage T4D with 2-hydroxy-5-nitrobenzyl bromide, a specific reagent for alkylating the indole ring of tryptophan residues, converts these particles from a cofactor-independent form to a cofactor-sensitive form. These treated T4D particles phenotypically resemble T4B particles in certain respects. Their ability to form plaques on minimal medium plates is increased by the addition of l-tryptophan and is inhibited by the addition of indole. In liquid medium, their rate of adsorption is dependent on the presence of the cofactor l-tryptophan. l-Tryptophan-requiring phage have been produced by in vitro assembly of treated tail-fiberless particles of a T4D amber mutant plus untreated tail fiber preparation. When treated tail fibers were used with untreated tail-fiberless particles, the newly assembled particles did not require cofactor. A model of the tail structure of all the T-even bacteriophages is presented which postulates that the active configuration of the tail fibers requires that there be either (i) an endogenous tryptophan residue of the phage particle itself or (ii) an exogenously added l-tryptophan molecule complexed with a specific tryptophan receptor site, most likely on the phage base plate.  相似文献   

20.
Precursors of the tail of bacteriophage λ have been detected by measurements of in vitro complementation activities and serum blocking activity in sucrose gradients of lysates defective in tail genes.On the basis of these measurements, a pathway for the assembly of the λ tail is proposed:The morphogenesis of the λ tail starts from the tail fiber (product of gene J) located at the distal end of the tail, and proceeds to the proximal end. Gene J by itself produces a 15 S structure with serum blocking activity but without any detectable in vitro complementation activity, which may be the least advanced precursor of the λ tail or an abortive product. Functions of genes J, I, K, L are required for the formation of a 15 S precursor that has in vitro complementation activities with J, I, K and L lysates and serum blocking activity. If the products of genes G and H act on the latter 15 S precursor, a 25 S precursor is made, but this precursor seems either to be in equilibrium with the 15 S precursor or to degrade easily into the 15 S precursor. Gene M has a function of stabilizing the 25 S precursor. After the action of gene M product, the 25 S precursor is ready to serve as a nucleus on which the product of gene V (the major tail protein) assembles. However, gene U product is also necessary at this step for the correct assembly of the major tail protein on the 25 S precursor. Without gene U product the assembly of the major tail protein does not stop at the correct length and a polytail is formed instead of a morphologically normal tail. Finally, gene Z product acts on the morphologically normal tail and makes it a biologically active tail. Without the action of gene Z product, the defective tail binds to a head and forms a phage-like particle which is only very weakly infectious. (The position of gene T in the pathway is not determined, because no sus mutant is available in gene T.)Two abnormal, less efficient pathways are also present in vitro. (1) If gene U product acts on a polytail in an U lysate, the polytail finally binds to a head and forms a phage particle with an extra long tail which is infectious to a small extent. (2) The function of gene K seems to be bypassed to some extent: K lysates accumulate particles which sediment as fast as normal phage and which are complemented by other tail lysates.  相似文献   

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