Metastable structures and refolding kinetics in hok mRNA of plasmid R1

J.H.A.  NAGEL a1, A.P.  GULTYAEV a1a2, K.  GERDES a3 and C.W.A.  PLEIJ a1c1
a1 Leiden Institute of Chemistry, Gorlaeus Laboratories, 2300 RA Leiden, The Netherlands
a2 Section Theoretical Biology and Phylogenetics, Institute of Evolutionary and Ecological Sciences, Leiden University, 2311 GP Leiden, The Netherlands
a3 Department of Molecular Biology, Odense University, Dk-5230, Odense M, Denmark


Programmed cell death by hok/sok of plasmid R1 and pnd/pndB of R483 mediates plasmid maintenance by killing of plasmid-free cells. It has been previously suggested that premature translation of the plasmid-mediated toxin is prevented during transcription of the hok and pnd mRNAs by the formation of metastable hairpins in the mRNA at the 5′ end. Here, experimental evidence is presented for the existence of metastable structures in the 5′ leader of the hok and pnd mRNAs in vitro. The kinetics of refolding from the metastable to the stable structure in the isolated fragments of the 5′ ends of both the hok and pnd mRNAs could be estimated, in agreement with the structural rearrangement in this region, as predicted to occur during transcription and mRNA activation. The refolding rates of hok and pnd structures are slow enough to allow for the formation of downstream hairpin structures during elongation of the mRNAs, which thereby helps to stabilize the metastable structures. Thus, the kinetic refolding parameters of the hok and pnd mRNAs are consistent with the proposal that the metastable structures prevent premature translation and/or antisense RNA binding during transcription.

(Received April 19 1999)
(Revised June 7 1999)
(Accepted July 29 1999)

Key Words: branch migration; kinetic traps; RNA rearrangements; RNA refolding; strand displacement; structure probing.

c1 Reprint requests to: C.W.A. Pleij, Leiden Institute of Chemistry, Gorlaeus Laboratories, Einsteinweg 55, 2300 RA Leiden, the Netherlands; e-mail: C.Pley@chem.LeidenUniv.nl.