EFFICIENCY AND FREQUENCY OF TRANSLATIONAL COUPLING BETWEEN THE BACTERIOPHAGE-T4 CLAMP LOADER GENES

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 f actors of DNA replication systems. The T4 sliding clamp comes to encir cle DNA via the ''clamp loader'' activity inherent in two other T4 pro teins: 44 and 62. These proteins assemble into a pentameric complex wi th a precise 4:1 stoichiometry of proteins 44 and 62, Previous work es tablished that T4 genes 44 and 62, which are directly adjacent on poly cistronic 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 d ifferent host cell backgrounds was accomplished hy quantitative immuno blotting. The efficiency of translational coupling was obtained by det ermining the in vivo levels of gp62 that were synthesized when its tra nslation was either coupled to or uncoupled from the upstream translat ion of gene 44, Levels of gp44 were also measured to determine the rel ative stoichiometry of synthesis and the percentage of gp44 translatio n that was transmitted across the intercistronic junction (coupling fr equency). The results indicated a coupling efficiency of similar to 85 % and a coupling frequency of similar to 25% between the 44-62 gene pa ir 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 abs olute requirement for the synthesis of gp62.