The Pathway for DNA Recognition and RNA Integration by a Group II Intron Retrotransposon  Yasunori Aizawa, Qing Xiang, Alan M. Lambowitz, Anna Marie Pyle  Molecular Cell  Volume 11, Issue 3, Pages 795-805 (March 2003) DOI: 10.1016/S1097-2765(03)00069-8 Copyright © 2003 Cell Press Terms and Conditions

Figure 1 Insertion of RNP into Duplex DNA (A) Conventional model for the first step of RNP insertion into duplex DNA. Lariat group II intron RNA (dark oval with tail) exists as an RNP with intron-encoded protein (light, surrounding oval). (B) Reaction products for RNP insertion into the top strand of duplex DNA. A representative gel for reaction of RNP (100 nM) with DNA duplex (0.1 nM, 5′ end labeled on top strand [5′P-3′P]) at varying times. For comparison, the products of insertion into 3′ end-labeled top strand DNA are also shown (far right lane). Reaction products include free 5′P (the 35 nt upstream product), RNA-3′P (the 905 nt lariat intron RNA ligated to 15 nts of downstream sequence), and 5′P-RNA-3′P (the product of complete integration). (C) The evolution of partial (closed circle) and full (open triangle) reverse-splicing products. The amplitudes of these reactions are 50% and 7%, respectively. Molecular Cell 2003 11, 795-805DOI: (10.1016/S1097-2765(03)00069-8) Copyright © 2003 Cell Press Terms and Conditions

Figure 2 Reversal of the Integration Reaction (A) Duplex sequences of the 50 nt integration substrate (1) and three reverse-integration substrates (2–4). An arrow indicates the normal intron insertion site on the top strand. Intron binding sequences (IBS) in the top strand are underlined. Substrate 3 is the same as 2 except for eight point mutations in the bottom strand that pairs with the IBS region (shadowed). Substrate 4 is the same as 5′P. (B) The products and relative efficiency of integration reversal. The substrate in this reaction (lane 1) is lariat RNA that is covalently attached to 3′P (Lariat RNA-3′P, labeled at the 3′ terminus). A small population of the RNA-3′P represents a nicked lariat structure (Linear RNA-3′P). The expected top strand product of integration reversal (5′P-3′P, equivalent to a forward splicing product) is shown (lane 2). When RNA-3′P is reacted with 2, one sees ligation of 5′P with 3′P, resulting in full length of the 50 nt top strand (8% efficiency, lane 3). Integration reversal of RNA-3′P with 3 and 4 is more efficient (51% and 53%; lanes 4 and 5, respectively). Molecular Cell 2003 11, 795-805DOI: (10.1016/S1097-2765(03)00069-8) Copyright © 2003 Cell Press Terms and Conditions

Figure 3 Determination of Kd and Active RNP Concentration Amplitudes (from curves of fraction P versus time) as a function of cold target DNA, plotted for three different concentrations of reconstituted RNP: 20 (closed circles), 60 (closed squares), and 100 (closed triangles) nM. The fit of each data set to Equation S6 results in the active RNP concentration and RNP-DNA Kd (see Supplementary Experimental Procedures and Table S1). Molecular Cell 2003 11, 795-805DOI: (10.1016/S1097-2765(03)00069-8) Copyright © 2003 Cell Press Terms and Conditions

Figure 4 Determining kfor, krev, and koff Using Pulse-Chase Experiments (A) The experimental scheme for the pulse-chase experiments. (B) Time course for accumulation of fraction P after addition of chase (arrow). A typical RNA insertion reaction (5.2 nM active RNP) with labeled target DNA (0.1 nM) (closed circles). The same reaction, but with a chase of unlabeled target DNA (100 nM) after 2 minutes of reaction (t1)(closed squares). (C) Pulse-chase experiments involving RNP (1.7 nM active), labeled substrate DNA (0.1 nM), and unlabeled substrate DNA (100 nM) at varying times prior to chase, where t1 = 1.5, 3, 5, 7.5, and 10.5 min, as indicated on inset. Each data set was fit to Equation S10 and are shown in Supplementary Table S2. Molecular Cell 2003 11, 795-805DOI: (10.1016/S1097-2765(03)00069-8) Copyright © 2003 Cell Press Terms and Conditions

Figure 5 Determination of kon Using Pulse-Chase Experiments (A) Conceptual plot of fraction P (total fraction ES and P) at the chase time (t2 = 0). Data plots obtained after the chase were fit to Equation S10 (black curve, see Supplementary Material). If the RNP-DNA complex did not dissociate after the chase, the kinetic profile of product formation would be dictated solely by the progress of the first phase (dotted curve). Amplitude of this theoretical curve would be the sum total of fraction P at the chase, FraP(t2 = 0), and the amplitude of this phase, m1. (B) Determination of kobs for the DNA binding step. As described in (A), fraction (ES + P) at various t1 was determined by pulse-chase experiments using two different concentrations of active RNP. Fraction (ES + P) was plotted versus t1 and fit with a single exponential. (C) kon and koff determination. The observed rate constants obtained from the fit of the data shown in (B) plotted versus active RNP concentration. Having fixed the y intercept at koff (0.005 min−1), the slope of the line is equivalent to kon (7.9 ± 0.3 × 107 M−1min−1). Molecular Cell 2003 11, 795-805DOI: (10.1016/S1097-2765(03)00069-8) Copyright © 2003 Cell Press Terms and Conditions

Figure 6 Kinetic Simulations of RNP Insertion The evolution of fraction product in the presence of 1.7 (A) or 14 nM (B) active RNP. Filled circles and squares represent the evolution of product from the first and second steps of insertion, respectively. Lines represent the simulation based on the mechanism in Scheme 2. Molecular Cell 2003 11, 795-805DOI: (10.1016/S1097-2765(03)00069-8) Copyright © 2003 Cell Press Terms and Conditions

Molecular Cell 2003 11, 795-805DOI: (10.1016/S1097-2765(03)00069-8) Copyright © 2003 Cell Press Terms and Conditions

Molecular Cell 2003 11, 795-805DOI: (10.1016/S1097-2765(03)00069-8) Copyright © 2003 Cell Press Terms and Conditions