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

Abstract

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.

Figure 1

The stable and metastable conformation of the hok⁷⁴ and pnd⁵⁸ 5′-end RNA fragments. The position of metastable hairpin I is indicated in red and the position of metastable hairpin II is indicated in green. (A) Stable structure and (B) metastable structure.

Figure 2

The kinetic refolding from the metastable to the stable conformation in the hok⁷⁴ RNA fragment. The metastable conformation was trapped by the heating/cooling cycle and the kinetics monitored at 260 nm in a UV spectrophotometer at 37°C in 950 mM NaCl and 50 mM Na cacodylate buffer, pH 7.2. The measured real-time curve (in black) and the first-order exponentially fitted curve is indicated in light blue (fitted parameters t_1/2 = 669 ± 1 s).

Figure 3

Comparison of refolding kinetics, using the heating/cooling and acid/alkaline cycle. (A) The UV absorbance measured curve, showing the refolding kinetics from the metastable to the stable conformation in the hok⁷⁴ RNA fragment at 37°C in 950 mM NaCl and 50 mM Na cacodylate buffer, pH 7.2, after the heating/cooling (in red) and acid/alkaline (in black) cycle and the fitted first-order kinetic curves [in light blue (fitted parameters t_1/2 = 566 ± 1 s) and in green (fitted parameters t_1/2 = 534 ± 2 s), respectively]. The dead times for the fitted curves are indicated with purple boxes (see also Fig. 2). (B) Measurement of refolding kinetics using the re-isolated (from the acid/alkaline cycle) hok⁷⁴ RNA fragment and used again in another acid/alkaline cycle. The measured curve (in black) was obtained at 37°C in 950 mM NaCl and 50 mM Na cacodylate buffer, pH 7.2 and fitted with a first-order exponential curve [in light blue (with the fitted parameters t_1/2= 521 ± 2 s)].

Figure 4

Refolding of the hok⁷⁴ RNA fragment at different temperatures. After applying the acid/alkaline cycle, the UV absorbance was measured at 260 nm at 27 (in blue), 32 (in red), 37 (in black) and 42°C (in green) in 1 M NaCl, pH 7.2.

Figure 5

The Årrhenius plots for the hok⁷⁴ (squares) and pnd⁵⁸ (circles) 5′-end RNA fragments.

Figure 6

Models for the possible refolding pathways from the metastable to the stable conformation, of the hok⁷⁴ and pnd⁵⁸ 5′-end RNA fragments. The bottom up or branch migration model is indicated in blue. The pseudoknot or strand displacement model is indicated in red. (A) Stable structure and (B) metastable structure.