The major head protein of bacteriophage T4 binds specifically to elongation factor Tu.

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.

Specific peptide-activated proteolytic cleavage of Escherichia coli elongation factor Tu.

Phage exclusion is a form of programmed cell death in prokaryotes in which death is triggered by infection with phage, a seemingly altruistic response that limits multiplication of the phage and its spread through the population. One of the best-characterized examples of phage exclusion is the exclusion of T-even phages such as T4 by the e14-encoded Lit protein in many Escherichia coli K-12 strains. In this exclusion system, transcription and translation of a short region of the major head coat protein gene late in phage infection activates proteolysis of translation elongation factor Tu (EF-Tu), blocking translation and multiplication of the phage. The cleavage occurs between Gly-59 and Ile-60 in the nucleotide-binding domain. In the present work, we show that a 29-residue synthetic peptide spanning the activating region of the major head coat protein can activate the cleavage of GDP-bound EF-Tu in a purified system containing only purified EF-Tu and purified Lit protein. Lit behaves as a bona fide enzyme in this system, cleaving EF-Tu to completion when present at substoichiometric amounts. Two mutant peptides with amino acid changes that reduce the activation of cleavage of EF-Tu in vivo were also greatly reduced in their ability to activate EF-Tu cleavage in vitro but were still able to activate cleavage at a high concentration. Elongation factor G, which has the same sequence at the cleavage site and a nucleotide-binding domain similar to EF-Tu, was not cleaved by this system, and neither was heat-inactivated EF-Tu, suggesting that the structural context of the cleavage site may be important for specificity. This system apparently represents an activation mechanism for proteolysis that targets one of nature's most evolutionarily conserved proteins for site-specific cleavage.