A pH-jump approach for investigating secondary structure refolding kinetics in RNA

TY - JOUR

T1 - A pH-jump approach for investigating secondary structure refolding kinetics in RNA

AU - Nagel, JHA

AU - Gultyaev, AP

AU - Oistamo, KJ

AU - Gerdes, K

AU - Pleij, CWA

PY - 2002

Y1 - 2002

N2 - 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 hok(74) and pnd(58) 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.

AB - 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 hok(74) and pnd(58) 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.

U2 - 10.1093/nar/gnf057

DO - 10.1093/nar/gnf057

M3 - Journal article

SN - 0305-1048

VL - 30

JO - Nucleic Acids Research

JF - Nucleic Acids Research

IS - 13

ER -