Volume 28, Issue 1, Pages 159-166 (October 2007) Do DEAD-Box Proteins Promote Group II Intron Splicing without Unwinding RNA?  Mark Del Campo, Pilar Tijerina, Hari Bhaskaran, Sabine Mohr, Quansheng Yang, Eckhard Jankowsky, Rick Russell, Alan M. Lambowitz  Molecular Cell  Volume 28, Issue 1, Pages 159-166 (October 2007) DOI: 10.1016/j.molcel.2007.07.028 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Unwinding of the 6 bp P1 Duplex (A) Gels showing unwinding of P1 in solution under standard conditions. Cartoons show the duplex and separated strand, with the asterisk indicating the radiolabel. (B and C) Unwinding of P1 in solution at 25°C by Mss116p WT (circles) or SAT/AAA (triangles) under standard (B) or splicing (C) conditions. Dissociation rates without protein are also shown (squares), and kcat/Km values are indicated. Both proteins also displayed P1 unwinding activity under splicing conditions at 30°C, but spontaneous helix dissociation was too fast to quantitatively measure enhancement by Mss116p (data not shown). (D) Unwinding of the P1 duplex linked to the ribozyme by Mss116p WT (circles) or SAT/AAA (triangles). Open and closed symbols are for reactions with or without saturating ATP-Mg2+ (2 mM), respectively. Molecular Cell 2007 28, 159-166DOI: (10.1016/j.molcel.2007.07.028) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 Unwinding of a 13 bp Duplex (A and B) Reactions with Mss116p WT and SAT/AAA, respectively. Gels show reactions with 400 nM Mss116p under splicing (left) and standard low-salt (right) conditions. Cartoons depict the duplex and separated strand as in Figure 1. Plots show the unwinding rate constant, calculated by including strand-annealing activity (Yang and Jankowsky, 2005), against protein concentration. Differences in the amount of RNA unwound in the steady state reflect that Mss116p's annealing activity is higher under the low-salt conditions (Halls et al., 2007). Data points are averages of multiple measurements, error bars indicate standard deviations, and solid lines are best fits by Hill binding curves. Kinetic parameters are shown below. Molecular Cell 2007 28, 159-166DOI: (10.1016/j.molcel.2007.07.028) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 Group II Intron Splicing (A and E) Splicing reactions with group II introns aI5γ and bI1, respectively, at 30°C for 120 min. Lane 1, RNA without incubation; lanes 2–10, reactions with indicated additions. P, precursor RNA; I-lar, intron lariat; I-lin, mix of linear intron and broken lariat; and E1-E2, ligated exons. (B, C, F, and G) Time courses of precursor RNA disappearance at 30°C in the presence or absence of the indicated concentrations of Mss116p WT or SAT/AAA plus 1 mM ATP-Mg2+. Data were fit with a single exponential. (D and H) Rate constant for precursor RNA disappearance as a function of protein concentration for aI5γ and bI1, respectively. kcat/Km values are shown. Rate constants represented by closed symbols are from the time courses in (B), (C), (F), and (G), whereas open symbols are from an independent experiment. Molecular Cell 2007 28, 159-166DOI: (10.1016/j.molcel.2007.07.028) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Group I Ribozyme Unfolding (A) Progress curves of unfolding with Mss116p WT or SAT/AAA at 800 or 1500 nM. (B) Observed rate constants for loss of native ribozyme as a function of Mss116p WT (circles) and SAT/AAA (triangles) concentration. kcat/Km values are shown. Molecular Cell 2007 28, 159-166DOI: (10.1016/j.molcel.2007.07.028) Copyright © 2007 Elsevier Inc. Terms and Conditions