Efficiency and Frequency of Translational Coupling between the Bacteriophage T4 Clamp Loader Genes

Genes 44 and 62 of bacteriophage T4 produce two proteins that are essential for the replication of the phage DNA (1). These proteins assemble into an oligomeric complex, gp44-gp62 (gp44/62), that possesses very low intrinsic ATPase activity. The ATPase activity of the gp44/62 complex is greatly stimulated upon its specific interaction with another T4-encoded protein, gp45 (14, 23). gp45 is the “sliding clamp” of the T4 system and is structurally homologous to the other known sliding clamps, namely, the β subunit of Escherichia coli and the eukaryotic proliferating cell nuclear antigen (20, 21). In vitro studies in a variety of laboratories have led to the consensus view that the role of gp44/62 during a T4 infection is to chaperone the gp45 protein to DNA (16, 30). Upon binding to DNA, the gp44/62 complex imparts a transient discontinuity to the ring-like structure of the T4 sliding clamp, resulting in the gp45 protein being topologically deposited onto double-stranded DNA (3, 22). That is, the gp44/62 complex is a “clamp loader.” Although it is presumed that in vivo this function is driven by the hydrolysis of ATP, the exact molecular mechanism by which this event occurs remains unknown. The role of gp44/62 as a clamp loader is catalytic (16). Once loaded onto DNA, the gp45 sliding clamp subsequently associates with the T4 DNA polymerase (gp43) to form the processive holoenzyme (9, 17, 31, 34). The T4 clamp loader is assembled upon the tight binding of one gp62 subunit to a gp44 tetramer (15, 35). The in vivo mechanism by which this strict 4:1 stoichiometry of the gp44/62 complex is maintained remains unknown. It is to be appreciated that the gp44/62 complex is similar in both sequence and function to the E. coli γ complex and eukaryotic RF-C (19, 28). Furthermore, the latter two clamp loaders are also composed of five subunits; however, the implications of the conservation in evolution of this pentameric stoichiometry have yet to be determined. The 44 and 62 genes occur in a region of the T4 genome clustered with other genes required for DNA replication. The genes of the T4 replication complex are arranged so that they may be transcribed as a cassette including genes 45, 44, 62, regA, and 43. They are expressed early in the infection cycle, and the transcripts are polycistronic (11). Transcription proceeds from early or middle promoters upstream of gene 45. There are strong consensus translation initiation regions (TIR) serving genes 45 and 44. The polycistronic message is subject to at least two known forms of translational regulation. One is repression due to direct binding of the RegA protein to the TIR of gene 44 (37). The second form of regulation is translational coupling (see references 6, 13, and 25 for reviews). This event occurs when translation of a distal gene on a polycistronic mRNA is strongly, or exclusively, dependent upon translation of a gene immediately upstream. In 1979, the laboratory of Karam initially provided evidence that the expression of gene 44 and that of gene 62 are translationally coupled (18). However, reflecting an inability to detect the low levels of gp62 produced, the precise degree of translational coupling was not determined by those investigators. In the current study, by employing specific antibodies and an extremely sensitive chemiluminescence detection protocol, we have quantitatively determined the levels of gp62 present in vivo during T4 infections. We have also examined the degree of translational coupling as a first step in determining how strict stoichiometry may be established for the gp44/62 complex.