Abstract
The k-turn is a commonly occurring structural motif that introduces a tight kink into duplex RNA. In free solution, it can exist in an extended form, or by folding into the kinked structure. Binding of proteins including the L7Ae family can induce the formation of the kinked geometry, raising the question of whether this occurs by passive selection of the kinked structure, or a more active process in which the protein manipulates the RNA structure. We have devised a single-molecule experiment whereby immobilized L7Ae protein binds Cy3-Cy5-labeled RNA from free solution. We find that all bound RNA is in the kinked geometry, with no evidence for transitions to an extended form at the millisecond timescale of the camera. Furthermore, real-time binding experiments provide no evidence for a more extended intermediate even at the earliest times, at a time resolution of 16 ms. The data support a passive conformational selection model by which the protein selects a fraction of RNA that is already in the kinked conformation, thereby drawing the equilibrium into this form.
Highlights
The kink-turn (k-turn) is a widespread structural motif found in many functional double-stranded RNA species that introduces a tight kink into the duplex axis with an included angle of close to 60 [1]
In addition to the role in the ribosome, protein binding to k-turns occurs as a critical initial step in the formation of the box C/D and H/ACA complexes that mediate site-specific modification of RNA in archaea and eukaryotes [9,10], and the 15.5 kDa protein binds U4 snRNA in the U4-U6.U5 tri-snRNP involved in Submitted September 27, 2012, and accepted for publication November 6, 2012
We have found that A. fulgidus L7Ae binds to H. marismortui Kt-7 with very high affinity, with Kd 1⁄4 10 pM, inducing the formation of the kinked geometry of the k-turn [3]
Summary
The kink-turn (k-turn) is a widespread structural motif found in many functional double-stranded RNA species that introduces a tight kink into the duplex axis with an included angle of close to 60 [1]. Most k-turns found in both ribosomal subunits are bound to different proteins [4,5]. These include L7Ae, which is bound to Kt-15 in the Haloarcula marismortui 50S ribosomal subunit [4]. In addition to the role in the ribosome, protein binding to k-turns occurs as a critical initial step in the formation of the box C/D and H/ACA complexes that mediate site-specific modification of RNA in archaea and eukaryotes [9,10], and the 15.5 kDa protein binds U4 snRNA in the U4-U6.U5 tri-snRNP involved in Submitted September 27, 2012, and accepted for publication November 6, 2012
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