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 transf er between enterobacterial cells is the FinOP system, traJ, a positive regulator of the transfer (tra) genes is controlled at the post-trans criptional level by two negative elements, finP and finO, FinP is a pl asmid-specific antisense RNA, whereas finO encodes a proteic co-repres sor which is not plasmid specific but exchangeable among F-like plasmi ds. We designed a traJ-lacZ test system that allowed us to monitor the effects of FinP and various FinP mutants on traJ expression, Furtherm ore, the introduction of finO into the test system enabled us to asses s 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 pl asmid, in the absence of FinO, repressed traJ expression six-fold, Whe n expressed from a pBR322-derived multicopy plasmid FinP repressed tra J expression approx, 2000-fold, This result unambiguously demonstrated that FinP is sufficient to repress traJ expression in a gene dosage-d ependent manner, Mutations of finP creating base exchanges either in l oop 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 lo op I-loop II mutation the repressor activity was almost completely los t, supporting the model that the first critical interaction between th e two RNA molecules occurs via 'kissing' of both loops of the RNAs, Ad dition of finO to the test system enhanced the repression of traJ expr ession by FinP by up to two orders of magnitude. This effect of FinO o n FinP activity in vivo might indicate that FinO, in addition to its f unction as an RNA stabilizer, promotes complex formation between the t arget mRNA and the antisense RNA, Such a function of FinO has recently been shown to exist in vitro (van Biesen and Frost (1994) Mol Microbi ol 14: 427-436).