THE ZINC COORDINATION SITE OF THE BACTERIOPHAGE-MU TRANSLATIONAL ACTIVATOR PROTEIN, COM

The bacteriophage Mu Com protein is a small ''zinc finger-like'' prote in that binds a specific site in com-mom operon mRNA and activates tra nslation of the mom open-reading-frame. Com contains six cysteine and five histidine residues that have the potential to form several altern ative zinc-finger-like motifs. We have used oligonucleotide site-direc ted mutagenesis to individually alter each of these amino acids (Cys t o Ser, and His to Asn or Gln) and tested the various forms of Com for their ability to function in vivo. We observed that mutation of any on e of the four N-terminal cysteine residues (Cys-6, 9, 26 or 29) result ed in loss of Com activity. The Com protein requires zinc in order to fold into its functional tertiary structure, as demonstrated by charac teristic H-1 nuclear magnetic resonance (NMR) chemical shifts. H-1 che mical shifts revert to random coil values in the presence of the metal chelator EDTA. The metal-binding specificity and thermal stability of Com also has been investigated using H-1 NMR. We report the use of Cd -113 NMR, H-1-Cd-113 heteronuclear spin-echo difference spectroscopy H SED and Zn extended X-ray absorption fine structure spectroscopy EXAFS to determine the zinc/protein stoichiometry as 1:1 and the ligand env ironment as tetrathiolate. Comparative NMR spectra of Com mutants C6S and C39S suggest position 6 is involved in zinc coordination, while po sition 39 is not metal-liganded. These studies indicate that the metal coordination, site of Corn is a four-cysteine complex, involving resi dues 9, 26 and 29.