Isao Katsura

Length determination in bacteriophage lambda tails

We have isolated viable mutants of bacteriophage lambda that have in-frame deletions in gene H, which codes for a minor tail protein. They produce correspondingly smaller but active gene H protein products and assemble shorter-tailed phage particles. The deficiency in tail length for each mutant corresponds to the calculated shortening of the gene H protein caused by the deletion. These results show that the H protein determines tail length and argue strongly for a scheme in which the H protein is a ruler or template that measures length during tail assembly.

Structure and assembly of bacteriophage lambda.

Bacteriophage λ has an icosahedral head with a radius of 30 nm and a flexible tail 150 nm long (Fig. lb; Kellenberger, 1961; Eiserling and Boy de la Tour, 1965; Kemp et al., 1968; Eiserling as quoted in Kellenberger and Edgar, 1971; Bayer and Bocharov, 1973; Mazza and Felluga, 1973; Harrison et al., 1973).

Morphogenesis of the tail of bacteriophage lambda: III. Morphogenetic pathway☆☆☆

Abstract 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.

Purification and characterization of the major protein and the terminator protein of the bacteriophage λ tail

Abstract Two proteins of the tail of bacteriophage λ were purified in an active form and characterized for the study of phage assembly. The terminator protein (product of gene U), which stops the polymerization of the major tail protein at the correct tail length, was purified from crude lysates. It exists as a globular monomer of 16,000 daltons in the absence of magnesium ions and as a ring-like hexamer in the presence of 20 mM MgSO4. It is a very acidic protein poor in lysine and lacking cysteine residues. Its secondary structure is rich in β-sheet and relatively poor in α-helix. Experiments using its antibody show that it is located at the proximal end of the tail. The major tail protein (product of gene V) was purified from dissociated tails or phage ghosts. This preparation was indistinguishable from the unassembled major tail protein in tail-defective lysates with respect to various properties except that the in vitro complementation activity was significantly lower. The gene V product consists of a polypeptide chain of about 25,000 daltons, which is rich in valine and threonine and which has no cysteine and no or an extremely small amount of histidine. Its secondary structure contains a large amount of random-coil, a relatively small amount of β-sheet, and very small amounts of α-helix. In solution it exists in a monomer-dimer equilibrium, and polymerizes only in the presence of the initiator for its assembly. The antibody against the major tail protein attaches all over the tubular part but not to the basal part of the tail.

Mechanism of length determination in bacteriophage lambda tails

The mechanism of length determination in bacteriophage lambda tails is discussed as a model for regulation in protein assembly systems. The lambda tail is a long flexible tube ending in a conical part and a single tail fiber. Its length is exactly determined in the sense that the number of major tail protein (gpV) molecules, which comprise more than 80% of the mass of the tail, is exactly the same in all tails. Assembly of gpV is regulated by the initiator complex, which contains the tail fiber and the conical part, and by the terminator protein gpU. There are two key points in the assembly of gpV with respect to length determination. (1) Assembly of gpV on the initiator pauses at the correct tail length. Binding of gpU to the tail only fixes the pause firmly. (2) When the tail length is too short, binding of gpU to tails is inhibited. Deletions and a duplication (both in frame) in gene H, which codes for one of the proteins in the initiator, result in production of phage particles with altered tail length. Moreover, the tail length is roughly proportional to the length of the mutated versions of gene H. This shows that the tail length is measured by the length of gene H protein (gpH), which seems to be approximately as long as the tail tube, if extended like a thread, according to secondary structure prediction (alpha-helices connected by other structures). Various pieces of evidence show that about six molecules of gpH are attached to the remaining portion of the initiator by the C-terminal part and folded into a somewhat compact form, while they are elongated as they are enclosed in the tail tube during assembly of gpV. Unlike interaction between the length-measuring genome RNA and the coat protein of tobacco mosaic virus, the major tail protein gpV does not bind specifically to the ruler protein gpH. Rather, gpH determines the tail length by inhibiting the binding of gpU to short tails and by signalling the pause when the correct tail length is attained.

Structure and function of the major tail protein of bacteriophage lambda: Mutants having small major tail protein molecules in their virion

Abstract Viable mutants of bacteriophage lambda having small major tail protein molecules in their virion have been isolated as pseudo-revertants of a defective prophage mutant ( defK244 ) in gene V , which codes for the major tail protein. According to deletion mapping, the defK244 mutation is located near the translation terminal of gene V , whereas some mappable reversion mutations leading to small major tail protein molecules map upstream to defK244 but still downstream to all the amber mutations tested. This suggests (if not proves) that the removable part is located at or near the carboxyl terminal of the major tail protein. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis and buoyant density measurements of the mutant phage particles show that as much as one-third of the major tail protein molecule can be removed without losing its capacity to maintain the total shape and infectivity of the phage particles. In the three-dimensional structure of the tail the removable part of the molecule exists as a protrusion at the outer part of the tail tube according to electron microscopy and hydrodynamic calculations based on sedimentation velocity experiments.

Morphogenesis of bacteriophage lambda tail. Polymorphism in the assembly of the major tail protein.

The major tail protein of bacteriophage lambda can polymerize into three different structures: (1) the tail, (2) the polytail, and (3) the polytube. They are characterized, respectively, as (1) a structure of exactly determined length having the tail tip, (2) a structure of undetermined length having the tail tip, and (3) a structure of undetermined length lacking the tail tip. This report shows that the polytube is made by aberrant initiation, whereas the polytail is made by incorrect termination of assembly. Genetic evidence suggests that the polymerization of the major tail protein (gene V product) into the polytube takes place on a pseudo-initiator, the formation of which is controlled by genes M and L . The assembly of both the tail and the polytail starts on the normal initiator which requires at least seven gene products for its formation. In this case, the elongation reaction pauses at the correct tail length even in the absence of the terminator protein (gene U product), and yields a tail precursor which we call “ U − -tail”. If the terminator protein attaches to the top of the U − -tail, it completes the termination of polymerization, and further action of the gene Z product makes it the tail. However, if more major tail protein molecules attach to the top of the U − -tail before the terminator protein attaches, the elongation reaction resumes and finally yields the polytail. Two abnormal reactions occur in the polytail pathway. (1) The gene Z product can act on the polytail without the action of the gene U product, and (2) the gene H product is released during the formation of the polytail.

Structure and inherent properties of the bacteriophage lambda head shell: IV. Small-head mutants☆☆☆

Missense mutants of bacteriophage lambda that produce small proheads were found among prophage mutants defective in the major head protein gpE. Measurements of the sedimentation coefficient and molecular weight of the small proheads showed that they have the T = 4 structure composed of 240 molecules of gpE instead of the wild-type T = 7 structure composed of 420 molecules of gpE. When the phage mutants were grown in groE mutants of Escherichia coli, they produced small unprocessed proheads, which contained a smaller number (about 60) of the core protein (gpNu3) molecules than normal unprocessed proheads, which contain about 180 molecules of gpNu3. This shows that the major head protein determines the size of not only the shell but also the core of unprocessed proheads. These mutants by themselves produce very few mature small-headed phage particles, partly because the lambda DNA molecule, whose cos sites are separated at a distance of 48,500 bases, is too long to be packaged into the small proheads. However, the small proheads can package shorter DNA in vivo and in vitro at somewhat reduced efficiency, if the length or a multiple of the length between the cos sites of the DNA is 13,000 to 19,000 bases.

Structure and inherent properties of the bacteriophage lambda head shell

Some mutations in the major capsid protein (gpE) of lambda phage can alter the size and shape of the head shell or block the pathway of head maturation. Previous studies on the classification of such mutants showed that there are at least five functional sites on the gpE molecule. In this study, we determined the amino acid exchanges by DNA sequencing to elucidate the molecular design of the form-determining multifunctional protein gpE. In addition, we characterized the mutated gpE molecules by two-dimensional gel electrophoresis and studied suppression patterns of amber mutants at 43 amino acid residues. Those mutations map at 19 amino acid residues at 22 bases, which are located in three regions, 40 to 91, 222 to 246, and 284 to 324 of the 341 amino acid residues of gpE. These regions seem to be important in the activity of gpE, since amber mutations in these regions are suppressed on the average by less species of suppressors than those outside these regions. The mutations having different phenotypes are not segregated from each other, while some mutations having the same phenotype are separated far apart in the primary structure. This suggests that the functional sites were formed during evolution after the folding pattern of the ancestral gpE polypeptide chain had been established. Many of the mutations are located at serine, glycine and proline residues in predicted beta-turns.

Structure and inherent properties of the bacteriophage lambda head shell: I. Polyheads produced by two defective mutants in the major head protein

New types of polyheads of bacteriophage lambda, produced by two defective mutants (def K13 and def K213; Katsura, 1976) in the major head protein, were studied by electron microscopy and optical diffraction, and compared with the normal polyheads consisting of non-mutated major head protein molecules. The new polyheads were found to be interesting for the following three reasons. 1. (1) They provide a good experimental system in which to study the changes in the quaternary structure due to small perturbations in the primary structure of protein molecules. The results of measurements of their pitch angle and width agreed with the rule of “constant near-equatorial lattice line curvature” proposed by Steven et al. (1976). This rule can be explained theoretically if one assumes that hypothetical ring-like nuclei are formed during the assembly of polyheads. 2. (2) Some of the polyheads produced by one of the mutants (def K213) have irregularities such as capping, widening, bending and branching. These irregularities seem to be due to the presence of a small number of pentamer(s) and/or heptamers(s) in the lattice consisting of hexamers. Theoretical predictions based on this assumption agree with the results. 3. (3) A one-sided image of the lambda head shell was obtained by optical filtering of the def K213 polyheads which have pitch angles that are different from 30 °. The filtered image revealed the arrangements of structural units in the capsomers.