9513979 Snyder Many plasmids and prophages exclude superinfecting phages by killing the host cell to prevent multiplication of the phage. The DNA element el4 is a defective prophage of E. coli K12 that excludes T4 and related phages by encoding a zinc metalloprotease that cleaves translation elongation factor Tu (EF-Tu) after infection. Cleavage of EF-Tu blocks cellular translation and prevents development of the phage. The protease is normally inactive, but is activated after infection by a short peptide determinant of only 25 amino acids internal to the major head protein of the infecting phage. Part of this grant is to begin to determine the mechanism of activation of the Lit protease which may serve as a prototype for signal transduction in other systems. The protease can be activated in crude extracts by the chemically synthesized peptide from the major head protein, making it possible to study the activation in vitro. This work will require purifying the protease to determine if any other factors are required, and determining whether the activating peptide binds to the protease or to EF-Tu. Clues to the mechanism of activation will come from an analysis of what amino acids in the protease are required for its activity and activation, and what amino acids in the peptide are required for the activation. Mutants in the protease and the peptide will be obtained both by site-specific mutagenesis and by directly selecting mutants defective in the activation. Other proteins may be cleaved by the protease since the amino acid sequence surrounding the site of cleavage of EF-Tu has been conserved throughout evolution and is also similar in other translation factors and in Gproteins in general. In addition, the role of the peptide coding region in the major head protein gene of T4 will be studied. This region of the major head protein gene seems to play a special regulatory role, greatly increasing the expression of downstream genes in transcriptional fusions. Experiments will establish whether thi s region is an transcription antiterminator, or acts by some other mechanism. If the region is an antiterminator, as seems most likely, the minimum sequence required for antiterminator activity will be determined, as well as the role of specific sequences and the function of translation in activating the antiterminator activity. Finally, the ability of this system to specifically inactivate Ef-Tu in vivo will be used to investigate the function of EF-Tu in the cell, including the proposed mechanism for the generation of ppGpp and a possible more direct role for EF-Tu in ribosomal RNA synthesis. Many types of cells make highly specific enzymes that kill cells in response to virus infection to prevent the spread of the virus to other cells. Components of the translation appparatus that synthesizes proteins is often the favored target of these enzymes because these are among the most highly conserved cellular constituents. Some of these enzymes are already finding applications in cell biology and biotechnology. Bacteria are known to make a number of enzymes that kill the cell in reponse to virus infection, but the molecular target of most of most of these enzymes is not understood. These studies should determine how the EF-Tu specific enzyme is activated by a peptide, anticipating that this mechanism will be used in other types of cells. These studies will also determine the potential of the EF-Tu specific protease in studying how cells regulate the expression of their genes. ??
