In vivo assembly of the tau-complex of the DNA polymerase III holoenzyme expressed from a five-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 Escherichia coli tau-complex on a single artificially constructed operon under the control of an inducible promoter. The proteins tau, delta, delta , chi, and psi overproduced from this artificial operon assemble efficiently in vivo, providing an efficient source of homogeneous tau-complex. The gamma subunit is a truncated form of tau that is produced by a translational frameshift. When protein expression was induced in bacterial strains containing the outer membrane protein T (OmpT) protease, tau was proteolyzed after lysis to a gamma-like protein, gammaP, and a peptide, C-tau, corresponding to the C terminus of tau. N-terminal sequencing of C-tau revealed a cleavage site between two lysines at positions 429 and 430 of tau. The deduced sequence of gammaP is, therefore, only two amino acids shorter than natural gamma. The proteolysis by OmpT was also shown directly by using purified OmpT and tau-complex in an in vitro reaction. A gammaP-complex and a mixed tau-gammaP-complex were purified from ompT+ cells. When the tau-complex proteins were overexpressed in ompT- bacteria, intact tau-complex lacking gammaP could be purified.
Pritchard
A E
AE
Department of Biochemistry, Biophysics, and Genetics and Graduate Program in Molecular Biology, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
Dallmann
H G
HG
McHenry
C S
CS
D001483:Base Sequence; D003001:Cloning, Molecular; D004257:DNA Polymerase II / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D009876:Operon; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D012697:Serine Endopeptidases / Q000378:metabolism
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D004257:DNA Polymerase II; D012697:Serine Endopeptidases; C057840:omptin outer membrane protease