Inhibition of protein synthesis from DNA replication genes by the product of gene 1, a dna gene of Bacillus subtilis phage .PHI.29.

Abstract

The DNA polymerase (Pol, 66kDa, product of gene 2) and the primer protein (PP, 31 kDa, product of gene 3 ) of Bacillus subtilis phage Φ29 are indispensable, together with the other phage-encoded DNA binding proteins, for the protein-primed DNA replication of Φ29. Immediately after infection, genes of these essential proteins are transcribed in a polycistronic fashion. For the further investigation of these essential proteins, genes 2 and 3 of Φ29 were cloned together into an Escherichia coli expression-vector system with T7 promoter. At the same time, gene 1, located downstream of gene 2, was incidentally cloned in the same segment with genes 2 and 3 due to the site of the restriction enzymes used. Though gene 1 belongs to dna genes, its function is still unclear. Upon induction of these cloned genes, we observed a very limited amount of Pol and PP in comparison to the product of gene 1 (gpl). To determine the reason for this large difference in gene expression between these three polycistronic genes, highly expressed gene, gene 1 was removed. As the deletion of gene 1, a reasonably high level of expression from the genes of both Pol and PP appeared. Efficient expression was also observed when gene 1 was substituted with its suppressor-sensitive allele, demonstrating that gpl affected the expression of its upstream genes 2 and 3 in E. coli. This evidence is consistent with the very small amount of both Pol and PP detected in the Φ29-infected cells of B. subtilis, and implies that gpl acts as a regulator of protein synthesis of the DNA replication genes.