Assembly of the bacteriophage T4 replication machine requires the acidic carboxy terminus of gene 32 protein.
The acidic carboxy-terminal 89-amino acid fragment of bacteriophage T4 gene 32 protein was expressed in Escherichia coli to high levels from an inducible plasmid construct. Infection of induced cells by wild-type T4 phage results in impaired phage DNA synthesis. The time at which DNA synthesis begins and the diminution in DNA synthesis rates correlate with the amount of carboxy-terminal peptide that accumulates intracellularly prior to infection. Correspondingly, when induced cells are infected with viable phage containing a small deletion near the carboxy-terminus of 32 protein (delta PR201), the inhibition of phage DNA synthesis was much more severe. The mutant 32 protein competes less well against overproduced wild-type acid peptide than does wild-type 32 protein. The purified acid peptide, when used as the attached ligand for affinity chromatography, binds several T4 proteins from phage-infected cells, including 43 protein (T4 DNA polymerase), Dda protein (a DNA helicase), and UvsX protein (a Rec-like recombination protein). Furthermore, at 50- to 100-fold molar excess of acid peptide over intact 32 protein, phage DNA synthesis was specifically inhibited at the initiation step in an in vitro 5-protein DNA replication experiment. We propose that one or more phage replication proteins are titrated as non-productive protein-protein complexes at a site away from the DNA template. This implies that the carboxy-terminal domain of 32 protein is involved in an obligate step of replication machine assembly when the protein is properly attached to ssDNA in the vicinity of a primer-template junction. The assembly defect we observe is strikingly similar to the repression, or "squelching", of the activity of certain eukaryotic transcriptional activators.
Hurley
J M
JM
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder 80309.
Chervitz
S A
SA
Jarvis
T C
TC
Singer
B S
BS
Gold
L
L
D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000235:genetics* / Q000502:physiology; D001483:Base Sequence; D002846:Chromatography, Affinity; D004261:DNA Replication / Q000235:genetics*; D004279:DNA, Viral / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D014764:Viral Proteins / Q000235:genetics*
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C060035:gp32 protein, Enterobacteria phage T4