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.

Isolation and evolutionary analysis of a RepFVIB replicon of the plasmid pSU212

“…Thus, our data are not contradictory with the hypothesis of 'ironing'. Moreover, the presence of the stop codon supports the idea that the protein itself is not necessary for the regulation as suggested by Wagner et al (1987).…”

“…This inhibition of binding would result in an enhanced expression of the RepA protein (Wagner et al, 1987).…”

Organization of the replication control region of plasmid ColIb-P9

“…The involvement of small RNA molecules in regulating gene expression has been reported in several plasmids, including IncFII (3,17,43,44) and pT181 (14). These RNAs have been shown to interact with mRNA of the target genes at their complementary regions and consequently to prevent expression of the respective genes at the translational level.…”

“…The nucleotide sequences of these small RNAs are complementary to parts of RNA molecules essential for replication. In ColEl (15,40,41), the small RNA termed RNAI inhibits formation of the primer for replication by binding to pre-primer RNAII. On the other hand, in IncFll (3,17,43,44) and pT181 (14), the small RNAs are thought to regulate the expression of genes encoding replication proteins. However, the precise mechanisms, especially those involved in the latter cases, remain to be investigated.…”

Role of countertranscript RNA in the copy number control system of an IncB miniplasmid

“…However, it should be pointed out that our failure to detect any promoter activity within the first 334 bp of pMU720 DNA indicates that a CopB-like protein is not encoded upstream of the RNA II promoter. Moreover, the absence of an open reading frame spanning the region complementary to the coding sequence of RNA I indicates that pMU720 does not encode a protein equivalent to the 7-kilodalton protein of the IncFII plasmids (37,55).…”