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.