Overexpression and purification of the primase domain of bacteriophage P4 gp alpha for structure determination by NMR

Abstract

Bacteriophage P4 is a temperate phage of E. coli and other Gram-negative prokaryotes. Its alpha gene encodes a 777-residue multifunctional protein, gpa, that is essential and sufficient for the initiation of P4 DNA replication. The gpa protein attaches to the P4 origin of replication, unwinds the DNA, and synthesizes a short strand of RNA which acts as a primer for the DNA polymerase of the host organism. These three activities, which in bacteria are carried out by three individual proteins, can be attributed to distinct gpa domains. The N-terminus of gpa constitutes the RNA polymerase, or primase, domain. We have overexpressed the gpa[1-373] primase domain in E. coli on minimal medium and are currently optimizing the purification procedure. It is intended to obtain the structure in solution of uniformly 13C,15N-labeled gpa[1-373] by ultra high-field 3D NMR spectroscopy. Primases of eubacteria and their phages and plasmids share sequence similarities, and comparison of a bacteriophage primase structure with the known structures of crystals of E. coli DnaG and P. furiosus primase domains could yield valuable insights into the mechanism of DNA-directed RNA polymerase activity.