SPECIFIC PEPTIDE-ACTIVATED PROTEOLYTIC CLEAVAGE OF ESCHERICHIA-COLI ELONGATION-FACTOR TU

Phage exclusion is a form of programmed cell death in prokaryotes in w hich death is triggered by infection with phage, a seemingly altruisti c response that limits multiplication of the phage and its spread thro ugh the population, One of the best-characterized examples of phage ex clusion is the exclusion of T-even phages such as T4 by the e14-encode d Lit protein in many Escherichia coil 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 o f translation elongation factor Tu (EF-Tu), blocking translation and m ultiplication of the phage, The cleavage occurs between Gly-59 and Ile -60 in the nucleotide-binding domain. In the present work, we show tha t 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 p rotein, Lit behaves as a bona fide enzyme in this system, cleaving EP- Tu to completion when present at substoichiometric amounts, Two mutant peptides with amino acid changes that reduce the activation of cleava ge of EF-Tu in vivo were also greatly reduced in their ability to acti vate EF-Tu cleavage in vitro but were still able to activate cleavage at a high concentration, Elongation factor G, which has the same seque nce at the cleavage site and a nucleotide-binding domain similar to EF -Tu, was not cleaved by this system, and neither was heat-inactivated EP-Tu, suggesting that the structural context of the cleavage site may be important for specificity, This system apparently represents an ac tivation mechanism for proteolysis that targets one of nature's most e volutionarily conserved proteins for site-specific cleavage.