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

摘要

The bacteriophage T4 DNA polymerase holoenzyme is composed of the core polymerase, gene product 43 (gp43), in association with the “sliding clamp” of the T4 system, gp45. Sliding clamps are the processivity factors of DNA replication systems. The T4 sliding clamp comes to encircle DNA via the “clamp loader” activity inherent in two other T4 proteins: 44 and 62. These proteins assemble into a pentameric complex with a precise 4:1 stoichiometry of proteins 44 and 62. Previous work established that T4 genes 44 and 62, which are directly adjacent on polycistronic mRNA molecules, are—to some degree—translationally coupled. In the present study, measurement of the levels (monomers/cell) of the clamp loader subunits during the course of various T4 infections in different host cell backgrounds was accomplished by quantitative immunoblotting. The efficiency of translational coupling was obtained by determining the in vivo levels of gp62 that were synthesized when its translation was either coupled to or uncoupled from the upstream translation of gene 44. Levels of gp44 were also measured to determine the relative stoichiometry of synthesis and the percentage of gp44 translation that was transmitted across the intercistronic junction (coupling frequency). The results indicated a coupling efficiency of ∼85% and a coupling frequency of ∼25% between the 44-62 gene pair during the course of infection. Thus, translational coupling is the major factor in maintaining the 4:1 stoichiometry of synthesis of the clamp loader subunits. However, coupling does not appear to be an absolute requirement for the synthesis of gp62.