The Lit protease in Escherichia coli K-12 strains induces cell death in response to bacteriophage T4 infection by cleaving translation elongation factor (EF) Tu and shutting down translation. Suicide of the cell is timed to the appearance late in the maturation of the phage of a short peptide sequence in the major head protein, the Gol peptide, which activates proteolysis. In the present work we demonstrate that the Gol peptide binds specifically to domains II and III of EF-Tu, creating the unique substrate for the Lit protease, which then cleaves domain I, the guanine nucleotide binding domain. The conformation of EF-Tu is important for binding and Lit cleavage, because both are sensitive to the identity of the bound nucleotide, with GDP being preferred over GTP. We propose that association of the T4 coat protein with EF-Tu plays a role in phage head assembly but that this association marks infected cells for suicide when Lit is present. Based on these data and recent observations on human immunodeficiency virus type 1 maturation, we speculate that associations between host translation factors and coat proteins may be integral to viral assembly in both prokaryotes and eukaryotes.