IN-VIVO ASSEMBLY OF THE TAU-COMPLEX OF THE DNA-POLYMERASE-III HOLOENZYME EXPRESSED FROM A 5-GENE ARTIFICIAL OPERON - CLEAVAGE OF THE TAU-COMPLEX TO FORM A MIXED GAMMA-TAU-COMPLEX BY THE OMPT PROTEASE

A plasmid was constructed that encodes all five subunits of the Escher ichia coli tau-complex on a single artificially constructed operon und er the control of an inducible promoter. The proteins tau, delta, delt a', chi, and psi overproduced from this artificial operon assemble eff iciently in vivo, providing an efficient source of homogeneous tau-com plex. The gamma subunit is a truncated form of tau that is produced by a translational frameshift. When protein expression was induced in ba cterial strains containing the outer membrane protein T (OmpT) proteas e, tau was proteolyzed after lysis to a gamma-like protein, gamma(P), and a peptide, C-tau, corresponding to the C terminus of tau. N-termin al sequencing of C-tau revealed a cleavage site between two lysines at positions 429 and 430 of tau. The deduced sequence of gamma(P) is, th erefore, only two amino acids shorter than natural gamma. The proteoly sis by OmpT was also shown directly by using purified OmpT and tau-com plex in an in vitro reaction. A gamma(P)-complex and a mixed tau-gamma (P)-complex were purified from ompT(+) cells. When the tau-complex pro teins were overexpressed in ompT(-) bacteria, intact tau-complex lacki ng gamma(P) could be purified.