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J Mol Biol. 1999 Jan 29;285(4):1457-73.

Degradation of FinP antisense RNA from F-like plasmids: the RNA-binding protein, FinO, protects FinP from ribonuclease E.

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  • 1Department of Biological Sciences, University of Alberta, Edmonton, Alberta, T6G 2E9, Canada.

Abstract

Transfer of F-like plasmids is regulated by the FinOP system, which controls the expression of traJ, a positive regulator of the transfer operon. F FinP is a 79 base antisense RNA, composed of two stem-loops, complementary to the 5' untranslated leader of traJ mRNA. Binding of FinP to the traJ leader sequesters the traJ ribosome binding site, preventing its translation and repressing plasmid transfer. The FinO protein binds stem-loop II of FinP and traJ mRNA and promotes duplex formation in vitro. FinO stabilizes FinP, increasing its effective concentration in vivo. To determine how FinO protects FinP from decay, the degradation of FinP was examined in a series of ribonuclease-deficient strains. Using Northern blot analysis, full-length FinP was found to be stabilized sevenfold in an RNase E-deficient strain. The major site of RNase E cleavage was mapped on synthetic FinP, to the single-stranded region between stem-loops I and II. A secondary site near the 5' end ( approximately 10 bases) was also observed. A GST-FinO fusion protein protected FinP from RNase E cleavage at both sites in vitro. Two duplexes between FinP and traJ mRNA were detected in an RNase III-deficient strain. The larger duplex resulted from extension of the FinP transcript at its 3' end, suggesting readthrough at the terminator that corresponds to FinP stem-loop II. A point mutant of finP (finP305; C30U) that is unable to repress traJ in the presence of FinO was also characterized. The pattern of RNase E digestion of finP305 RNA differed from FinP, and GST-FinO did not protect finP305 RNA from cleavage in vitro. The half-life of finP305 RNA decreased more than tenfold in vivo, such that the steady-state levels of finP305 RNA, in the presence of FinO, were insufficient to significantly reduce the level of traJ mRNA available for translation, allowing derepressed levels of transfer.

Copyright 1999 Academic Press.

PMID:
9917389
[PubMed - indexed for MEDLINE]
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