Dissecting the multistep reaction pathway of an RNA enzyme by single-molecule kinetic "fingerprinting"

@article{citeulike:1320420, abstract = {Edited by Robert J. Silbey, Massachusetts Institute of Technology, Cambridge, MA, and approved April 4, 2007 (received for review November 30, 2006)Single-molecule FRET is a powerful tool for probing the kinetic mechanism of a complex enzymatic reaction. However, not every reaction intermediate can be identified via a distinct FRET value, making it difficult to fully dissect a multistep reaction pathway. Here, we demonstrate a method using sequential kinetic experiments to differentiate each reaction intermediate by a distinct time sequence of FRET signal (a kinetic "fingerprint"). Our model system, the two-way junction hairpin ribozyme, catalyzes a multistep reversible RNA cleavage reaction, which comprises two structural transition steps (docking/undocking) and one chemical reaction step (cleavage/ligation). Whereas the docked and undocked forms of the enzyme display distinct FRET values, the cleaved and ligated forms do not. To overcome this difficulty, we used Mg2+ pulse-chase experiments to differentiate each reaction intermediate by a distinct kinetic fingerprint at the single-molecule level. This method allowed us to unambiguously determine the rate constant of each reaction step and fully characterize the reaction pathway by using the chemically competent enzyme-substrate complex. We found that the ligated form of the enzyme highly favors the docked state, whereas undocking becomes accelerated upon cleavage by two orders of magnitude, a result different from that obtained with chemically blocked substrate and product analogs. The overall cleavage reaction is rate-limited by the docking/undocking kinetics and the internal cleavage/ligation equilibrium, contrasting the rate-limiting mechanism of the four-way junction ribozyme. These results underscore the kinetic interdependence of reversible steps on an enzymatic reaction pathway and demonstrate a potentially general route to dissect them. 10.1073/pnas.0610597104}, author = {Liu, Shixin and Bokinsky, Gregory and Walter, Nils G. and Zhuang, Xiaowei }, citeulike-article-id = {1320420}, doi = {10.1073/pnas.0610597104}, journal = {PNAS}, keywords = {fret, proteins, sms}, month = {May}, pages = {0610597104+}, posted-at = {2007-05-23 07:06:22}, priority = {4}, title = {Dissecting the multistep reaction pathway of an RNA enzyme by single-molecule kinetic "fingerprinting"}, url = {http://dx.doi.org/10.1073/pnas.0610597104}, year = {2007} }

TY - JOUR ID - citeulike:1320420 L3 - citeulike-article-id:1320420 TI - Dissecting the multistep reaction pathway of an RNA enzyme by single-molecule kinetic "fingerprinting" JF - PNAS SP - 0610597104 N2 - Edited by Robert J. Silbey, Massachusetts Institute of Technology, Cambridge, MA, and approved April 4, 2007 (received for review November 30, 2006)Single-molecule FRET is a powerful tool for probing the kinetic mechanism of a complex enzymatic reaction. However, not every reaction intermediate can be identified via a distinct FRET value, making it difficult to fully dissect a multistep reaction pathway. Here, we demonstrate a method using sequential kinetic experiments to differentiate each reaction intermediate by a distinct time sequence of FRET signal (a kinetic "fingerprint"). Our model system, the two-way junction hairpin ribozyme, catalyzes a multistep reversible RNA cleavage reaction, which comprises two structural transition steps (docking/undocking) and one chemical reaction step (cleavage/ligation). Whereas the docked and undocked forms of the enzyme display distinct FRET values, the cleaved and ligated forms do not. To overcome this difficulty, we used Mg2+ pulse-chase experiments to differentiate each reaction intermediate by a distinct kinetic fingerprint at the single-molecule level. This method allowed us to unambiguously determine the rate constant of each reaction step and fully characterize the reaction pathway by using the chemically competent enzyme-substrate complex. We found that the ligated form of the enzyme highly favors the docked state, whereas undocking becomes accelerated upon cleavage by two orders of magnitude, a result different from that obtained with chemically blocked substrate and product analogs. The overall cleavage reaction is rate-limited by the docking/undocking kinetics and the internal cleavage/ligation equilibrium, contrasting the rate-limiting mechanism of the four-way junction ribozyme. These results underscore the kinetic interdependence of reversible steps on an enzymatic reaction pathway and demonstrate a potentially general route to dissect them. 10.1073/pnas.0610597104 KW - fret KW - proteins KW - sms AU - Liu, Shixin AU - Bokinsky, Gregory AU - Walter, Nils AU - Zhuang, Xiaowei PY - 2007/05/11/ UR - http://dx.doi.org/10.1073/pnas.0610597104 ER -