J. Mol. Biol. (1984) 180, 283-300 A Functional Domain of Bacteriophage k Terminase for Prohead Binding SUSAN FRACKMAN, DEBORAH A. SIEGELE AND MICHAEL FEISS Department of Microbiology, College of Medicine University of Iowa, Iowa City, IA 52242, U.S.A. (Received 12 April 1984) Terminase is a multifunetional protein complex involved in DNA packaging during bacteriophage 2 assembly. Terminase is made of gpNul and gpA, the products of the phage 2 Nul and A genes. Early during DNA packaging terminase binds to 2 DNA to form a complex called complex I. Terminase is required for the binding of proheads by complex I to form a DNA : terminase : prohead complex known as complex II. Terminase remains associated with the DNA during encapsidation. The other known role for terminase in packaging is the production of staggered nicks in the DNA thereby generating the cohesive ends. Lambdoid phage 21 has cohesive ends identical to those of 2. The head genes of 2 and 21 show partial sequence homology and are analogous in structure, function and position. The terminases of 2 and 21 are not interchangeable. At least two actions of terminase are involved in this specificity: (1) DNA binding; (2) prohead binding. The 1 and 2 genes at the left end of the 21 chromosome were identified as coding for the 21 terminase, gpl and gp2 are analogous to gpNul and gpA, respectively. We have isolated a phage, ).-21 hybrid 33, which is the product of a crossover between ~l and 21 within the terminase genes. 2-21 hybrid 33 DNA and terminase have phage 21 packaging specificity, as determined by complementation and helper packaging studies. The terminase of 2-21 hybrid 33 requires 2 proheads for packaging. We have determined the position at which the crossover between 2 DNA and 21 DNA occurred to produce the hybrid phage. ~t-21 hybrid 33 carries the phage 21 1 gene and a hybrid phage 2/A gene. Sequencing of A-21 hybrid 33 DNA shows that it encodes a protein that is homologous at the earboxy terminus with the 38 amino acids of the carboxy terminus of 2 gpA; the remainder of the protein is homologous to gp2. The results of these studies define a specificity domain for prohead binding at the carboxy terminus of gpA. 1. Introduction The packaging of bacteriophage 2 DNA involves specific interactions that bring DNA and the empty protein shell together. The DNA substrate for packaging is a linear multichromosomal length DNA, called a concatemer. Concatemers are produced, at late times after ~ infection, primarily by rolling-circle replication (reviewed by Furth & Wickner, 1983). The empty protein shells, called proheads, into which the DNA is packaged are the product of an assembly pathway that requires 2 head proteins and host proteins (reviewed by Georgopoulos et al., 1983). 283 0022-2836/84/340283-18 $03.00/0 © 1984 Academic Press Inc. (London) Ltd.