Autoregulation of phage P22 scaffolding protein synthesis

During the formation of each P22 head, about 250 molecules of the scaffolding protein coassaemble with and dictate correct assembly of the coat protein into proper shell structures. Near the time the DNA is inserted inside the coat protein shell, all of the scaffolding protein molecules leave the structure intact, and participate in subsequent rounds of prohead assembly. All genes involved in particle assembly, including the scaffolding protein gene, map within the late operon, and are exclusively transcribed from the expression of other late genes. In vivo experiments have generated a model in which unassembled scaffolding protein negatively regulates its own synthesis at a posttranscriptional level, but lacks this activity when assemble in proheads. In order to test this model, the scaffolding protein coat protein gene were cloned under the control of the lac promoter, and expressed in a DNA-dependent translation reaction in vitro. Addition of physiological levels of purified scaffolding protein to this reaction resulted in specific depression of scaffolding protein synthesis, relative to the synthesis of coat protein and also the tail protein which was encoded by a separate plasmid. These results directly demonstrated scaffolding protein synthesis in autoregulated and that scaffolding protein is the only phage protein required for this autoregulation. The cloned P22 restriction fragment used in this experiment must contain all the sequences require for regulation of scaffolding protein synthesis in vitro. To define more precisely which sequences are required for regulation of scaffolding protein gene expression, plasmids encoding scaffolding protein ?-galactosidase hybrid proteins were constructed. These plasmids were tested for regulation of hybrid protein synthesis by unassembled scaffolding protein vivo. Scaffolding protein gene sequences upstreams of coden 63 were sufficient of regulation ofhybrid protein synthesis. However, sequences upstream of the 5'-end of the cloned template in the in vitro experiments are also required for regulation in this assay. Experiments to resole this apparent discrepancy should increase our understanding of autoregulation of scaffolding protein gene expression.