Jonathan King

Polypeptides of the tail fibres of bacteriophage T4

Abstract We have identified the products of four of the six genes involved in bacteriophage T4 tail fibre assembly by sodium dodecyl sulphate-acrylamide gel electrophoresis of tail fibre mutant lysates and particles purified from them. Two large polypeptides, a 150,000 molecular weight species which is the product of gene 34 (P34), and a 120,000 molecular weight species which is the product of gene 37 (P37), are the major structural components of the fibres. Two smaller polypeptides, the products of genes 38 and 57, act in the conversion of the large structural polypeptide chains into morphological and antigenic half fibres. P38, molecular weight 26,000, does not appear to be a structural protein of the phage. In its absence, P37 is synthesized but remains unassembled. P57 plays a pleiotropic role in phage assembly: in its absence, P37 and P34 are both synthesized, but neither is assembled into fibres, and P12, a 60,000 molecular weight protein of the baseplate, is not incorporated into baseplates. The state of these unassembled polypeptide chains from 38 - and 57 - lysates can be distinguished from their state in wild-type lysates by two criteria: (a) they are soluble in sodium dodecyl sulphate at room temperature, whereas normal fibres and phages require heating for solubilization, and (b) they are concentrated in the low-speed pellet fractions of the lysates, suggesting that they are either aggregated, or bound to the cell envelope. A gene 36 amber mutation depressed the synthesis of P37 and a gene 37 amber mutation depressed the synthesis of P38, suggesting that these three genes are cotranscribed. These findings allow the formulation in greater detail of the early stages of the fibre assembly pathway.

Cyanobacterial photosynthesis in the oceans: the origins and significance of divergent light-harvesting strategies

Prochlorococcus and Synechococcus are abundant unicellular cyanobacteria and major participants in global carbon cycles. Although they are closely related and often coexist in the same ocean habitat, they possess very different photosynthetic light-harvesting antennas. Whereas Synechococcus and the majority of cyanobacteria use phycobilisomes, Prochlorococcus has evolved to use a chlorophyll a(2)/b(2) light-harvesting complex. Here, we present a scenario to explain how the Prochlorococcus antenna might have evolved in an ancestral cyanobacterium in iron-limited oceans, resulting in the diversification of the Prochlorococcus and marine Synechococcus lineages from a common phycobilisome-containing ancestor. Differences in the absorption properties and cellular costs between chlorophyll a(2)/b(2) and phycobilisome antennas in extant Prochlorococcus and Synechococcus appear to play a role in differentiating their ecological niches in the ocean environment.

Structure of epsilon15 bacteriophage reveals genome organization and DNA packaging/injection apparatus

The critical viral components for packaging DNA, recognizing and binding to host cells, and injecting the condensed DNA into the host are organized at a single vertex of many icosahedral viruses. These component structures do not share icosahedral symmetry and cannot be resolved using a conventional icosahedral averaging method. Here we report the structure of the entire infectious Salmonella bacteriophage epsilon15 (ref. 1) determined from single-particle cryo-electron microscopy, without icosahedral averaging. This structure displays not only the icosahedral shell of 60 hexamers and 11 pentamers, but also the non-icosahedral components at one pentameric vertex. The densities at this vertex can be identified as the 12-subunit portal complex sandwiched between an internal cylindrical core and an external tail hub connecting to six projecting trimeric tailspikes. The viral genome is packed as coaxial coils in at least three outer layers with ∼90 terminal nucleotides extending through the protein core and the portal complex and poised for injection. The shell protein from icosahedral reconstruction at higher resolution exhibits a similar fold to that of other double-stranded DNA viruses including herpesvirus, suggesting a common ancestor among these diverse viruses. The image reconstruction approach should be applicable to studying other biological nanomachines with components of mixed symmetries.

Mechanism of head assembly and DNA encapsulation in Salmonella phage P22: I. Genes, proteins, structures and DNA maturation

Abstract The functions of ten known late genes are required for the intracellular assembly of infectious particles of the temperate Salmonella phage P22. The defective phenotypes of mutants in these genes have been characterized with respect to DNA metabolism and the appearance of phage-related structures in lysates of infected cells. In addition, proteins specified by eight of the ten late genes were identified by sodium dodecyl sulfate/polyacrylamide gel electrophoresis; all but two are found in the mature phage particle. We do not find cleavage of these proteins during morphogenesis. The mutants fall into two classes with respect to DNA maturation; cells infected with mutants of genes 5, 8, 1, 2 and 3 accumulate DNA as a rapidly sedimenting complex containing strands longer than mature phage length. 5− and 8− lysates contain few phage-related structures. Gene 5 specifies the major head structural protein; gene 8 specifies the major protein found in infected lysates but not in mature particles. 1−, 2− and 3− lysates accumulate a single distinctive class of particle (“proheads”), which are spherical and not full of DNA, but which contain some internal material. Gene 1 protein is in the mature particle, gene 2 protein is not. Cells infected with mutants of the remaining five genes (10, 26, 16, 20 and 9) accumulate mature length DNA. 10− and 26− lysates accumulate empty phage heads, but examination of freshly lysed cells shows that many were initially full heads. These heads can be converted to viable phage by in vitro complementation in concentrated extracts. 16− and 20− lysates accumulate phage particles that appear normal but are non-infectious, and which cannot be rescued in vitro. From the mutant phenotypes we conclude that an intact prohead structure is required to mature the virus DNA (i.e. to cut the overlength DNA concatemer to the mature length). Apparently this cutting occurs as part of the encapsulation event.

Assembly of the tau of bacteriophage T4

Abstract The formation of bacteriophage T4 has been studied by characterizing the phage components accumulating in cells infected—under restrictive conditions—with mutants blocked at different stages of the assembly process. Three structures which appear to be intermediates in tail assembly have been isolated by centrifugation: baseplates (S20,w ≅ 80 s), core-baseplates (S20,w ≅ 80 s), and core-baseplates with surrounding sheath (S20,w ≅ 130 s). The functions of genes 19, 48 and 54 are required for the conversion of the baseplate to the core-baseplate. The functions of genes 3, 15 and 18 are required for the formation of the sheath. Gene 18 codes for a major structural protein of the sheath. Genes 3 and 15 specify products required for the stabilization and completion of the sheath. Tail assembly is sequenced; the baseplate is completed first and the core forms on the baseplate. The gene 18 product polymerizes on the core-baseplate and then the 3 and 15 gene products fix the sheath subunits in the polymerized form. After sheath formation, a segment appears to be added to the core at the terminus of the sheath, permitting subsequent attachment of the head. The tail fibers attach only after the particle formed by head-tail union has been acted upon by the gene 9 product. Particles which have not been acted upon by the gene 11 or 12 product adsorb to bacteria but do not kill them. Electron microscopic observations on the state of phage heads in mutant lysates are also presented. Mutations in three genes result in the accumulation of head membranes empty of DNA. Phage heads and phage tails are formed independently of each other.

Nucleation and growth phases in the polymerization of coat and scaffolding subunits into icosahedral procapsid shells

The polymerization of protein subunits into precursor shells empty of DNA is a critical process in the assembly of double-stranded DNA viruses. For the well-characterized icosahedral procapsid of phage P22, coat and scaffolding protein subunits do not assemble separately but, upon mixing, copolymerize into double-shelled procapsids in vitro. The polymerization reaction displays the characteristics of a nucleation limited reaction: a paucity of intermediate assembly states, a critical concentration, and kinetics displaying a lag phase. Partially formed shell intermediates were directly visualized during the growth phase by electron microscopy of the reaction mixture. The morphology of these intermediates suggests that assembly is a highly directed process. The initial rate of this reaction depends on the fifth power of the coat subunit concentration and the second or third power of the scaffolding concentration, suggesting that pentamer of coat protein and dimers or trimers of scaffolding protein, respectively, participate in the rate-limiting step.

Backbone structure of the infectious ε15 virus capsid revealed by electron cryomicroscopy

A half-century after the determination of the first three-dimensional crystal structure of a protein, more than 40,000 structures ranging from single polypeptides to large assemblies have been reported. The challenge for crystallographers, however, remains the growing of a diffracting crystal. Here we report the 4.5-Å resolution structure of a 22-MDa macromolecular assembly, the capsid of the infectious epsilon15 (ε15) particle, by single-particle electron cryomicroscopy. From this density map we constructed a complete backbone trace of its major capsid protein, gene product 7 (gp7). The structure reveals a similar protein architecture to that of other tailed double-stranded DNA viruses, even in the absence of detectable sequence similarity. However, the connectivity of the secondary structure elements (topology) in gp7 is unique. Protruding densities are observed around the two-fold axes that cannot be accounted for by gp7. A subsequent proteomic analysis of the whole virus identifies these densities as gp10, a 12-kDa protein. Its structure, location and high binding affinity to the capsid indicate that the gp10 dimer functions as a molecular staple between neighbouring capsomeres to ensure the particle’s stability. Beyond ε15, this method potentially offers a new approach for modelling the backbone conformations of the protein subunits in other macromolecular assemblies at near-native solution states.

Mechanism of head assembly and DNA encapsulation in Salmonella phage P22: II. Morphogenetic pathway

Abstract We have identified and characterized structural intermediates in phage P22 assembly. Three classes of particles can be isolated from P22-infected cells: 500 S full heads or phage, 170 S empty heads, and 240 S “proheads”. One or more of these classes are missing from cells infected with mutants defective in the genes for phage head assembly. By determining the protein composition of all classes of particles from wild type and mutant-infected cells, and examining the time-course of particle assembly, we have been able to define many steps in the pathway of P22 morphogenesis. In pulse-chase experiments, the earliest structural intermediate we find is a 240 S prohead; it contains two major protein species, the products of genes 5 and 8 . Gene 5 protein (p5) is the major phage coat protein. Gene 8 protein is not found in mature phage. The proheads contain, in addition, four minor protein species, PI, P16, P20 and PX. Similar prohead structures accumulate in lysates made with mutants of three genes, 1, 2 and 3 , which accumulate uncut DNA. The second intermediate, which we identify indirectly, is a newly filled (with DNA) head that breaks down on isolation to 170 S empty heads. This form contains no P8, but does contain five of the six protein species of complete heads. Such structures accumulate in lysates made with mutants of two genes, 10 and 26 . Experiments with a temperature-sensitive mutant in gene 3 show that proheads from such 3 − infected cells are convertible to mature phage in vivo , with concomitant loss of P8. The molecules of P8 are not cleaved during this process and the data suggest that they may be re-used to form further proheads. Detailed examination of 8 − lysates revealed aberrant aggregates of P5. Since P8 is required for phage morphogenesis, but is removed from proheads during DNA encapsulation, we have termed it a scaffolding protein, though it may have DNA encapsulation functions as well. All the experimental observations of this and the accompanying paper can be accounted for by an assembly pathway, in which the scaffolding protein P8 complexes with the major coat protein P5 to form a properly dimensioned prohead. With the function of the products of genes 1, 2 and 3 , the prohead encapsulates and cuts a headful of DNA from the concatemer. Coupled with this process is the exit of the P8 molecules, which may then recycle to form further proheads. The newly filled heads are then stabilized by the action of P26 and gene 10 product to give complete phage heads.

Crystal cataracts: Human genetic cataract caused by protein crystallization

Several human genetic cataracts have been linked recently to point mutations in the γD crystallin gene. Here we provide a molecular basis for lens opacity in two genetic cataracts and suggest that the opacity occurs because of the spontaneous crystallization of the mutant proteins. Such crystallization of endogenous proteins leading to pathology is an unusual event. Measurements of the solubility curves of crystals of the Arg-58 to His and Arg-36 to Ser mutants of γD crystallin show that the mutations dramatically lower the solubility of the protein. Furthermore, the crystal nucleation rate of the mutants is enhanced considerably relative to that of the wild-type protein. It should be noted that, although there is a marked difference in phase behavior, there is no significant difference in protein conformation among the three proteins.

Scaffolding protein regulates the polymerization of P22 coat subunits into icosahedral shells in vitro

Coat and scaffolding subunits derived from P22 procapsids have been purified in forms that co-assemble rapidly and efficiently into icosahedral shells in vitro under native conditions. The half-time for this reaction is approximately five minutes at 21 degrees C. The in vitro reaction exhibits the regulated features observed in vivo. Neither coat nor scaffolding subunits alone self-assemble into large structures. Upon mixing the subunits together they polymerize into procapsid-like shells with the in vivo coat and scaffolding protein composition. The subunits in the purified coat protein preparations are monomeric. The scaffolding subunits appear to be monomeric or dimeric. These results confirm that P22 procapsid formation does not proceed through the assembly of a core of scaffolding, which then organizes the coat, but requires copolymerization of coat and scaffolding. To explore the mechanisms of the control of polymerization, shell assembly was examined as a function of the input ratio of scaffolding to coat subunits. The results indicated that scaffolding protein was required for both initiation of shell assembly and continued polymerization. Though procapsids produced in vivo contain about 300 molecules of scaffolding, shells with fewer subunits could be assembled down to a lower limit of about 140 scaffolding subunits per shell. The overall results of these experiments indicate that coat and scaffolding subunits must interact in both the initiation and the growth phases of shell assembly. However, it remains unclear whether during growth the coat and scaffolding subunits form a mixed oligomer prior to adding to the shell or whether this occurs at the growing edge.