Measurement of ATP utilization in RNA unwinding and RNA chaperone activities by DEAD-box helicase proteins

Abstract

RNA helicase proteins perform coupled reactions in which cycles of ATP binding and hydrolysis are used to drive local unwinding of double-stranded RNA (dsRNA). For some helicases in the ubiquitous DEAD-box family, these local unwinding events are integral to folding transitions in structured RNAs, and thus these helicases function as RNA chaperones. An important measure of the efficiency of the helicase-catalyzed reaction is the ATP utilization value, which represents the average number of ATP molecules hydrolyzed during RNA unwinding or a chaperone-assisted RNA structural rearrangement. Here we outline procedures that can be used to measure the ATP utilization value in RNA unwinding or folding transitions. As an example of an RNA folding transition, we focus on the refolding of the Tetrahymena thermophila group I intron ribozyme from a long-lived misfolded structure to its native structure, and we outline strategies for adapting this assay to other RNA folding transitions. For a simple dsRNA unwinding event, the ATP utilization value provides a measure of the coupling between the ATPase and RNA unwinding activities, and for a complex RNA structural transition it can give insight into the scope of the rearrangement and the efficiency with which the helicase uses the energy from ATPase cycles to promote the rearrangement.

Keywords: ATPase kinetics; CYT-19; DEAD-box protein; Double-stranded RNA; RNA chaperone; RNA helicase; RNA misfolding.

Fig. 1.

RNA binding and unwinding by DEAD-box helicase proteins. (A), Structural views of ssRNA binding by the helicase core, as shown for Mss116 (Del Campo & Lambowitz, 2009b). The ssRNA (purple) binds to Mss116 (blue) in a crimped conformation, incompatible with a standard duplex geometry. Also shown are a bound ATP analog (orange) and a coordinated Mg²⁺ ion (green). (B) ATP-dependent RNA unwinding by DEAD-box helicases. In the dominant pathway, highlighted in yellow, the ATP-bound form of the helicase binds dsRNA and produces local unwinding (starting from top left; ATP is indicated by a red triangle). This unwinding reaches completion, resulting in the dissociation of one of the strands, producing the ssRNA-bound form shown in panel A. ATP hydrolysis and release of P_i weaken binding of ssRNA (bound ADP is indicated by a purple circle), resulting in ssRNA dissociation (bottom right). Thus, this pathway results in complete helix unwinding with hydrolysis of one ATP. Less populated pathways produce ATPase activity without complete duplex unwinding (top two rows, counterclockwise) and complete duplex unwinding without ATP hydrolysis (left column, top to bottom).

Fig. 2.

Results of ATP utilization measurement in RNA unwinding by the DEAD-box helicase CYT-19 (Chen et al., 2008). (A) RNA unwinding substrate and labeling strategy. The RNA helix (red) was appended to a flanking DNA helix, which was shown to increase CYT-19 activity (Tijerina, Bhaskaran, & Russell, 2006). In the pulse phase, the RNA helix was formed by adding labeled CCCUCUA₅ (labeling indicated by asterisk), and unwinding of this helix was trapped in the chase phase by excess unlabeled CCCUCUA₅. Results are shown for RNA unwinding measurements (B) and ATPase measurements (C). For both sets of experiments, CYT-19 was 2 μM and the duplex was 0.5 μM. For ATPase measurements, excess of the substrate strand was present (1 μM). For RNA unwinding measurements, this strand was increased to 5 μM in panel C, which was shown to increase the signal for RNA unwinding without changing the observed rate constant. Conditions for all measurements were 25 °C, 10 mM Mg²⁺, and 50 μM ATP-Mg²⁺. These measurements gave an ATP utilization value of 1.1 ± 0.1.

Fig. 3.

Experimental workflows for measurement of ATP utilization during helicase-promoted RNA unwinding. Workflows are shown for ATPase (A) and RNA unwinding (B) rate measurements. Asterisks indicate radioactively labeled components (ATP or RNA).

Fig. 4.

Experimental workflows for measurement of ATP utilization during a helicase-promoted RNA folding transition. Workflows are shown for ATPase (A), RNA refolding (B), and combined ATPase and refolding (C) rate measurements. Asterisks indicate radioactively labeled components (ATP and/or ribozyme substrate oligonucleotide). In (C), the concentrations of folding quench components should be adjusted to be 80% lower than those listed in Section 3.3.2 (step 3) to account for the slightly larger volume required to provide sufficient sample for both TLC and gel analysis.

Fig. 5.

Determination of the ATP utilization value for ribozyme refolding. (A) The initial-rate method. Left: To obtain the ATP hydrolysis rate associated with refolding, the initial ATPase rates (slopes of linear fits) are determined during refolding (black; M) and in the presence of the pre-folded native ribozyme (grey; N). All panels depict simulated data based on previous results for CYT-19 and the Tetrahymena ribozyme (Jarmoskaite, Tijerina, & Russell, 2021). To narrow down the number of productive ATP hydrolysis events, for the Tetrahymena ribozyme, we subtract the background ATPase activity seen with N ribozyme from the ATPase rate measured during the refolding of M ribozyme (see notes in Section 3.4 for the underlying assumptions). Right: The initial refolding rate is determined both in the presence (+) and absence (−) of helicase and ATP, and the spontaneous rate is subtracted. The ATP utilization value is calculated using Eq. 4. (B) The full time-course approach. Left: The ATPase activity is recorded until the completion of the refolding reaction, i.e., until at least ~90% of the ribozyme has reached the native state (based on the refolding data). The same time points as those taken in the refolding reaction (black) are collected in the presence of pre-folded native ribozyme (grey). At each time point, the difference in the amount of hydrolyzed ATP is calculated between the two time courses (arrows) and the resulting values are plotted (right). The amplitude of the resulting curve (arrow) corresponds to the total amount of productive ATP hydrolysis. Division by the ribozyme concentration yields the number of ATPs hydrolyzed per ribozyme.