Comparison of the assembly of the bacteriophage T4 clamp loader complex (gp44/62) expressed in a cis versus trans genomic configuration.
Proper formation of the bacteriophage T4 DNA polymerase holoenzyme requires a wide spectrum of protein-protein and protein-DNA interactions among the DNA polymerase gp43, the sliding clamp gp45, and gp44/62, the clamp loader complex (CLC). The 44 and 62 proteins associate to form a tight complex maintained in a 4:1 ratio. The 44 and 62 genes are adjacent to each other on the T4 genome, are cotranscribed, and are translationally coupled. It has been suggested that translational coupling may play a role in the formation of the clamp loader complex and may control its stoichiometry. To examine the effect of coupling on the assembly of the complex, expression in trans of genes 44 and 62 was accomplished by cotransforming Escherichia coli with compatible, inducible plasmid vectors. A gp44/62 complex could be purified from such cells. The complex assembled in trans exhibited stoichiometry and ATPase activity identical to native complex. Burst sizes were determined to gauge the efficiency of clamp loader complex formation. When gp44 was supplied by a plasmid and gp62 was supplied by the T4 genome, complex formation was as efficient as in wild-type virus. However, when gp62 was supplied by plasmid and gp44 was supplied by the T4 genome, efficiency of complex formation was decreased. This decrease in the efficiency of complex formation was temperature dependent, being more pronounced at higher temperatures. At higher temperatures, a larger proportion of gp62 expressed from the plasmid was found to be present in an insoluble form. The decrease in efficiency of complex formation correlated to a decrease in solubility of the gene 62 protein.
Chemistry Department, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin, 53201-0413, USA.
D M
DM
Janzen
M Y
MY
Torgov
S N
SN
Abbott
M K
MK
Reddy
D017122:Bacteriophage T4; D001483:Base Sequence; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D012995:Solubility; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D019065:Virus Assembly
D014764:Viral Proteins; C074855:gene 44 protein, Enterobacteria phage T4; C088659:gp62 protein, bacteriophage T4; D004259:DNA-Directed DNA Polymerase