Translational regulation of M13 gene II protein by its cognate single‐stranded DNA binding protein

Abstract

To unravel the mechanism by which the single‐stranded DNA binding protein encoded by gene V of the filamentous phage M13 regulates the synthesis of its cognate DNA replication protein encoded by gene II, an in vivo test system has been developed. The system consists of two recombinant plasmids with compatible replication origins. One plasmid contains M13 gene V under the control of the inducible araB promoter of Salmonella typhimurium. The other plasmid contains a fusion gene, whose expression is dependent upon the M13 gene‐II‐promoter and which consists of the 5′ end of M13 gene II and the 5′‐truncated β‐galactosidase gene of Escherichia coli. Induction of the synthesis of wild‐type gene V protein by arabinose resulted in a specific reduction of both the β‐galactosidase activity and the amount of fusion protein produced. These specific inhibitory effects were not observed when the synthesis of the fusion protein was studied in the presence of an amber mutant of gene V. Comparison of the relative concentrations of the fusion protein mRNAs, as present in arabinose‐induced and noninduced cells, provided solid and direct evidence for the conclusions made in earlier publications, that gene V protein exerts its regulatory effect at the level of translation. Since the transcript of the fusion gene only contains the first 74 nucleotides of gene II mRNA, it is furthermore concluded that these nucleotides are already sufficient for gene V protein to exert its regulatory effect. Copyright © 1990, Wiley Blackwell. All rights reserved