Head Assembly Sequence and Host Proteins

The Head Assembly Sequence and Host Proteins

Head precursors of lambda can be isolated by centrifugation and can thereby be studied. The first consists of pE, pNu3, pB and pC (Fig. 21.16). Without pE, nothing forms, and without pNu3, only large amorphous shapes appear. The formation of this structure also requires that about 12 molecules of the pB protein be polymerized. This is done with the assistance of the host proteins GroEL and GroES. The pB protein ultimately forms the connector between head and tail.

In the head maturation process there is both cleavage of polypeptide chains and fusion of chains. For example, some molecules of pB are cleaved and some molecules of pE are fused to protein pC. The final head is formed by the addition of lambda DNA, its cleavage to unit-length molecules containing the 12-base single-stranded cos ends, and the addition of pD on an equimolar basis to pE. Proteins pW and pF_II prepare the head for attachment of the tail. One of the functions of such a protein is to act as an adapter between the hexagonal tail and the pentagonal vertex to which the tail is attached.

The protein pNu3 occupies a role predicted for many other proteins but thus far found rather infrequently. It appears to be a structural protein and is used during the assembly of the phage particle but is not present in the assembled phage particle. It is synthesized in high quantities and forms a part of the scaffolding during formation of the particle. In the case of lambda, this protein is cleaved after a single use, but in some other phage the analogous protein is used more than once.

The GroEL-GroES protein complex is a member of the class of proteins that assists the formation of complex protein structures. Often these proteins possess ATPase activities. Some disaggregate oligomers that have formed from hydrophobic interactions or prevent their for-mation. Others assist the molten globule state to perform final rear-rangements of secondary structure elements. The protein DnaK that functions in assembling the replication complex of lambda along with the phage P protein and the host DnaB protein, also participates in the lambda capsid maturation reactions. Mutants with altered GroE protein

Figure 21.16 Maturation scheme of lambda heads.

frequently fail to support the growth of not only phage lambda but also related phage and even phage T4. In addition to failing to cleave pB, they do not package DNA. Mutants of lambda that overcome the groE defect can be isolated. These are usually found to be altered in the lambda E gene. Some of the rarer groE mutations can be overcome by compensating mutations in the lambda B gene. These results strongly suggest that GroE protein touches the pE and pB proteins during the maturation of the phage.

The groE mutation joins the list of other cellular mutations that may block maturation of the phage. In addition to groE, there are also the groP mutations in the host cell, which block DNA replication of thephage, and groN mutations which make the phage appear to be N defective. The groP mutations lie within a subunit of DNA polymerase. Some groN mutations lie in the RNA polymerase and can be overcome by a compensating mutation in the phage N gene.