Control of replication of plasmid R1: translation of the 7k reading frame in the RepA mRNA leader region counteracts the interaction between CopA RNA and CopT RNA

Abstract

Replication of IncFII plasmids is regulated through the expression of a gene, repA. The RepA protein is rate-limiting for initiation of replication. The main negative control is exerted by a countertranscript, CopA RNA, that binds to the complementary region of the RepA mRNA, thereby inhibiting the formation of the RepA protein. The target region for CopA RNA, CopT, is located upstream of the RepA coding region. An open reading frame for a putative 7k protein overlaps the CopT sequence. Here we show by using lacZ fusions that the 7k gene is expressed. We constructed a translation start mutation in order to abolish formation of the 7k protein. This resulted in a 10-fold decrease in repA expression. The 7k protein produced in trans did not reverse this effect, so the 7k protein per se does not control expression of repA. However, translation of the 7k coding sequence must influence CopA/CopT RNA recognition, since ribosomes will transiently disrupt the target hairpin. We propose here a novel mechanism that affects the level of gene expression: the 7k region of the RepA mRNA is a leader sequence that is involved in expression of the downstream gene; translation of the 7k region competes with a negative control system involving RNA-RNA interaction.