Mechanochemistry of Transcription Termination Factor Rho Joshua L. Adelman, Yong-Joo Jeong, Jung-Chi Liao, Gayatri Patel, Dong-Eun Kim, George Oster, Smita S. Patel  Molecular Cell  Volume 22, Issue 5, Pages 611-621 (June 2006) DOI: 10.1016/j.molcel.2006.04.022 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 The Rho Hexamer (A) Open structure of Rho (PDB 1PVO) with a modeled RNA strand bound to the primary binding sites courtesy of Emmanuel Skordalakes and James Berger. Alternating subunits of the hexamer are shown in blue and yellow, and the hypothetical configuration of the RNA strand is shown in red. This structure most likely represents an initiation conformation, which is not competent to undergo ATP-driven translocation. (B) Structural detail showing the secondary site RNA binding Q loop, the nucleotide binding Walker A (P loop) and B motifs, and the arginine finger from the adjacent subunit. The R loop in this persepective is obscured by the Q loop. The RNA in the secondary site is shown as a cartoon circle because the exact interactions with the secondary binding site are unknown. (C) Cartoon showing the two stress loops that coordinate the progression of ATP hydrolysis cycles in each subunit. Molecular Cell 2006 22, 611-621DOI: (10.1016/j.molcel.2006.04.022) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Rho ATPase Inhibition and ADP·BeFx Binding (A) Rho (100 nM) was incubated with polyC (136 nM) in the Rho buffer, and ATP (1 mM) spiked with [α-32P]ATP was added to initiate the reaction. After 15–60 s time intervals at 18°C, reactions were stopped with 2 M formic acid and analyzed by PEI-cellulose TLC. The inhibitors were added at 0.5 mM concentrations with the ATP, except for NaF that was present at 2.5 mM concentration. (B) Steady-state binding of [BeFx]·ADP to transcription terminator Rho in the presence (circles) and absence (crosses) of polyC RNA. Solid lines represent least-squares fit by hyperbolic equation. Molecular Cell 2006 22, 611-621DOI: (10.1016/j.molcel.2006.04.022) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Time Trajectories of Chase-Time ATP Binding Experiments and Steady-State Amplitudes (A) The total [ADP] in solution and bound to Rho after initiation of ATPase activity. Time trajectories are shown for 1, 6, 50, 100, and 200 μM ATP. The data points are least-squares fit by equation 5. (B) Steady-state values of ADP production obtained from fitting the burst amplitude in equation 5. This quantity gives a lower bound on the number of ATPs initially bound to each hexamer. Molecular Cell 2006 22, 611-621DOI: (10.1016/j.molcel.2006.04.022) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 [ADP] Production and [Pi] Release (A) Time trajectories of acid-quench experiments tracking [ADP] production. Simulated trajectories are shown as solid lines and match the experimental data in both transient and steady-state phases. (B) PBP-MDCC monitored Pi release trajectory. Measurements are made for the same initial [ATP] as in the acid-quench experiments. The solid lines are fits to the kinetic model. (C) Comparison of Pi release and ADP production for a representative initial [ATP] (300 μM). Solid lines are the same simulation fittings as in (A) and (B). (D) Delay between ATP hydrolysis and Pi release for several initial conditions. The solid line is the mean delay over all concentrations. Molecular Cell 2006 22, 611-621DOI: (10.1016/j.molcel.2006.04.022) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 ATPase Activity of Mixed Mutant Hexamers Deficient in RNA Binding The steady-state ATPase activity was measured for a mixture of T286A and wild-type Rho. ATPase activity decreases as a function of increasing mutant subunits in solution. The solid line shows the predicted behavior for the sequential power stroke mechanism, whereas the dashed line shows the predicted behavior of a random power stroke model. Molecular Cell 2006 22, 611-621DOI: (10.1016/j.molcel.2006.04.022) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 The Sequential Mechanism of ATPase Powered Translocation Rho is shown “unwrapped” and viewed from inside the ring. (A) RNA is bound to subunit 1 in the NT∗ state, indicated by the black circle. Other subunits in the hexamer can be in different states, but at steady-state conditions, neighboring subunits are likely to be in the product, DP state because Pi release is rate limiting. (B) Binding of ATP at the RNA bound subunit drives a power stroke during the NT∗ → NT transition. The stress caused by tight binding of ATP radiates out from the binding site asymmetrically (indicated by the shading gradient) and is felt strongly by the adjacent subunit, 2. This stress helps the release of Pi and ADP at 2. RNA is only weakly bound by the subunit 2 Q-loop, indicated by the open circle. (C) Upon docking ATP into the now empty site in subunit 2, that site becomes competent to bind RNA (solid circle). The power stroke at the preceding subunit has brought the RNA into close proximity with B's secondary RNA binding site, allowing thermal fluctuations of the strand to allow attachment (T∗ → NT∗). When both subunits are attached to the RNA, a stress loop forms (dashed triangle), prompting a conformational change in the subunit 2 ATP binding site that optimally aligns the catalytic arginine finger permitting hydrolysis. (D) The hydrolysis of ATP in subunit 1 decreases its affinity for RNA (open circle). Release of RNA at 2 leaves the hexamer in the same conformation as (A) with the RNA shifted one subunit to the right. From this position, the cycle repeats, allowing Rho to sequentially translocate RNA through its central channel. Molecular Cell 2006 22, 611-621DOI: (10.1016/j.molcel.2006.04.022) Copyright © 2006 Elsevier Inc. Terms and Conditions

(1) Molecular Cell 2006 22, 611-621DOI: (10.1016/j.molcel.2006.04.022) Copyright © 2006 Elsevier Inc. Terms and Conditions