A Genetic analysis of bacteriophage lambda prohead assembly in vitro

Abstract

Phage lambda DNA can be packaged into proheads to generate full heads in vitro. Upon addition of tails and other gene products to the cell-free assembly system, infectious phage are formed. This system provides a convenient assay to measure biologically active proheads. Thus, it has been shown that biologically active proheads are not produced in wild-type cells infected with phage mutants in genes B, C, Nu3 and E, nor in groE mutant cells infected with wild-type phage. However, proheads can be assembled in vitro upon incubation of a mixture of λ Nu3 − and λ E − infected-cell extracts. This complementation reaction has been analyzed using extracts prepared from wild-type or groE − cells infected with phage carrying amber mutations in one or two prohead genes. The conclusion from this analysis can be summarized as follows. 1. (1) Among all the possible combinations of extracts of cells infected with single amber mutants, the E − + Nu3 − combination is the only one that gives substantial complementation in vitro. 2. (2) gpC † † The prefix gp stands for “gene product”. Therefore, gpC, etc., mean: products of gene C, etc. can be contributed by either member of the complementing pair of extracts. 3. (3) An E − B − extract will not complement a Nu3 − extract. 4. (4) Active gpB can only be derived from a Nu3 + extract, and/or active gpNu3 can only be derived from a B + extract. It is, therefore, probable that gpB and gpNu3 interact in vivo before gpE is polymerized. 5. (5) The lack of in vitro complementation exhibited by certain pairs of mutant extracts can be explained conveniently by postulating that essential gene products (such as gpE) are consumed in the assembly of abnormal capsid structures in vivo. 6. (6) The B − Nu3 − extract (gpE and gpC donor) does not require the function of the host groE gene product to provide complementing activity. 7. (7) groE function is essential for the production of the complementing activity in E − C − extracts (gpB and gpNu3 donor). 8. (8) If gpB and gpNu3 are synthesized in the absence of wild-type groE function, the defect in complementing activity cannot be repaired by the addition of groE + cell extracts. It thus appears that groE functions very early during prohead assembly, prior to the polymerization of the coat protein gpE.