A pH-jump approach for investigating secondary structure refolding kinetics in RNA.
It has been shown that premature translation of the plasmid-mediated toxin in hok/sok of plasmid R1 and pnd/pndB of plasmid R483 is prevented during transcription of the hok and pnd mRNAs by the formation of metastable hairpins at the 5'-end of the mRNA. Here, an experimental approach is presented, which allows the accurate measurement of the refolding kinetics of the 5'-end RNA fragments in vitro without chemically modifying the RNA. The method is based on acid denaturation followed by a pH-jump to neutral pH as a novel way to trap kinetically favoured RNA secondary structures, allowing the measurement of a wide range of biologically relevant refolding rates, with or without the use of standard stopped-flow equipment. The refolding rates from the metastable to the stable conformation in both the hok74 and pnd58 5'-end RNA fragments were determined by using UV absorbance changes corresponding to the structural rearrangements. The measured energy barriers showed that the refolding path does not need complete unfolding of the metastable structures before the formation of the final structures. Two alternative models of such a pathway are discussed.
Nagel
J H A
JH
Leiden Institute of Chemistry, Gorlaeus Laboratories, Einsteinweg 55, 2300 RA Leiden, The Netherlands.
Gultyaev
A P
AP
Oistämö
K J
KJ
Gerdes
K
K
Pleij
C W A
CW
D001426:Bacterial Proteins / Q000235:genetics; D001427:Bacterial Toxins; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009695:Nucleic Acid Renaturation; D010957:Plasmids / Q000235:genetics; D012313:RNA; D012329:RNA, Bacterial / Q000737:chemistry*; D013696:Temperature
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D029968:Escherichia coli Proteins; C087025:Hok protein, E coli; D012329:RNA, Bacterial; C087026:Sok antisense RNA, E coli; C087023:pndA protein, E coli; D012313:RNA