The FinOP repressor system of plasmid R1: analysis of the antisense RNA control of traJ expression and conjugative DNA transfer.

A key determinant of the frequency of IncF plasmid-mediated DNA transfer between enterobacterial cells is the FinOP system. traJ, a positive regulator of the transfer (tra) genes is controlled at the post-transcriptional level by two negative elements, finP and finO. FinP is a plasmid-specific antisense RNA, whereas finO encodes a proteic co-repressor which is not plasmid specific but exchangeable among F-like plasmids. We designed a traJ-lacZ test system that allowed us to monitor the effects of FinP and various FinP mutants on traJ expression. Furthermore, the introduction of finO into the test system enabled us to assess the function of FinO in the interaction of FinP with its target, the traJ mRNA. In this test system, FinP, expressed from a single-copy plasmid, in the absence of FinO, repressed traJ expression six-fold. When expressed from a pBR322-derived multicopy plasmid FinP repressed traJ expression approx. 2000-fold. This result unambiguously demonstrated that FinP is sufficient to repress traJ expression in a gene dosage-dependent manner. Mutations of finP creating base exchanges either in loop I or loop II of the two stem-loop structures of the antisense RNA led to a dramatic decrease in the repressor activity. In a combined loop I-loop II mutation the repressor activity was almost completely lost, supporting the model that the first critical interaction between the two RNA molecules occurs via 'kissing' of both loops of the RNAs. Addition of finO to the test system enhanced the repression of traJ expression by FinP by up to two orders of magnitude. This effect of FinO on FinP activity in vivo might indicate that FinO, in addition to its function as an RNA stabilizer, promotes complex formation between the target mRNA and the antisense RNA. Such a function of FinO has recently been shown to exist in vitro (van Biesen and Frost (1994) Mol Microbiol 14: 427-436).

Differential mRNA decay within the transfer operon of plasmid R1: identification and analysis of an intracistronic mRNA stabilizer.

Processing of the transfer operon mRNA of the conjugative resistance plasmid R1-19 results in the accumulation of stable traA mRNAs. The stable traA transcripts found in vivo have identical 3' ends within downstream traL sequences, but vary at their 5' ends. The 3' ends determined coincide with the 3' base of a predicted large clover-leaf-like RNA secondary structure. Here we demonstrate that this putative RNA structure, although part of a coding sequences, stabilizes the upstream traA mRNA very efficiently. We also show that the 3' ends of the stable mRNAs are formed posttranscriptionally and not by transcription termination. Half-life determinations reveal the same half-lives of 13 +/- 2 min for the traA mRNAs transcribed from hybrid lac-traAL-cat test plasmids, the R1-19 plasmid, and the F plasmid. Protein expression experiments demonstrate that the processed stable traA mRNA is translationally active. Partial deletions of sequences corresponding to the predicted secondary structure within the traL coding region drastically reduce the chemical and functional half-life of the traA mRNA. The results presented here unambiguously demonstrate that the proposed secondary structure acts as an efficient intracistronic mRNA stabilizer.

Gene 19 of plasmid R1 is required for both efficient conjugative DNA transfer and bacteriophage R17 infection.

F-like plasmids require a number of genes for conjugation, including tra operon genes and genes traM and traJ, which lie outside the tra operon. We now establish that a gene in the "leading region," gene 19, provides an important function during conjugation and RNA phage infection. Mutational inactivation of gene 19 on plasmid R1-16 by introduction of two nonpolar stop codons results in a 10-fold decrease in the conjugation frequency. Furthermore, infection studies with the male-specific bacteriophage R17 revealed that the phage is not able to form clear plaques in Escherichia coli cells carrying an R1-16 plasmid with the defective copy of gene 19. The total number of cells infected by phage R17 is reduced by a factor of 10. Both the conjugation- and infection-attenuated phenotypes caused by the defective gene 19 can be complemented in trans by introducing gene 19 alleles encoding the wild-type protein. Restoration of the normal phenotypes is also possible by introduction of the pilT gene encoded by the unrelated IncI plasmid R64. Our functional studies and similarities of protein 19 to proteins encoded by other DNA transfer systems, as well as the presence of a conserved motif in all of these proteins (indicative for a putative muramidase activity) suggest that protein 19 of plasmid R1 facilitates the passage of DNA during conjugation and entry of RNA during phage infection.

Expression of gene 19 of the conjugative plasmid R1 is controlled by RNase III.

Specific cleavage of mRNAs by RNase III has been shown to control the expression of several Escherichia coli genes. We show here that the expression of gene 19 of the conjugative resistance plasmid R1 is controlled in its expression by the same endoribonuclease. In vivo studies revealed that a DNA fragment of 150 nucleotides including a perfect 22 nucleotide inverted repeat in the gene 19 coding region is responsible for the low expression of the gene both at the protein and the RNA levels. By using a translational gene 19-lacZ fusion in isogenic RNase III+ and RNase III- strains we could identify RNase III as the key element in the down-regulation of gene 19 expression. The sequencing of in vitro generated and RNase III-digested transcripts confirmed the in vivo studies and revealed the exact positions of the RNase III cleavage sites within the coding part of the gene 19 transcript. The in vitro determined RNase III cleavage of gene 19 mRNA was confirmed by in vivo primer extension analysis. Finally, we could show that an exchange of three nucleotides within the RNase III recognition site abolished RNase III cleavage in vitro.