Sequence determinants of the folding properties of box C/D kink-turns in RNA

Abstract

Folding properties differ markedly between kink-turns (k-turns) that have different biological functions. While ribosomal and riboswitch k-turns generally fold into their kinked conformation on addition of metal ions, box C/D snoRNP k-turns remain completely unfolded under these conditions, although they fold on addition of L7Ae protein. Sequence elements have been systematically exchanged between a standard ribosomal k-turn (Kt-7) that folds on addition of metal ions, and a box C/D kturn. Folding was studied using fluorescence resonance energy transfer and gel electrophoresis. Three sequence elements each contribute in an approximately additive manner to the different folding properties of Kt-7 and box C/D k-turns from archaea. Bioinformatic analysis indicates that k-turn sequences evolve sequences that suit their folding properties to their biological function. The majority of ribosomal and riboswitch k-turns have sequences allowing unassisted folding in response to the presence of metal ions. In contrast, box C/D k-turns have sequences that require the binding of proteins to drive folding into the kinked conformation, consistent with their role in the assembly of the box C/D snoRNP apparatus. The rules governing the influence of sequence on folding properties can be applied to other standard k-turns to predict their folding characteristics.

The role of RNA structure in translational regulation by L7Ae protein in archaea

“…Some, but importantly not all, k-turns become stabilized on addition of metal ions (Goody et al 2004;Liu and Lilley 2007). Whether or not a given k-turn folds in response to metal ions depends upon its sequence, and we have found that two sequence elements act as key determinants of this behavior (McPhee et al 2014;Ashraf et al 2017). Protein binding provides an alternative way to stabilize the folded k-turn.…”

“…the absence of protein binding. We have previously studied this in some depth (McPhee et al 2014;Ashraf et al 2017), and have identified two critical sequence elements that determine the ability of a k-turn to fold in response to the presence of metal ions.…”

“…The −1b:−1n sequence. Recently we have found that the −1b:−1n sequence that precedes the bulge can have a strong influence on ion-induced folding (Ashraf et al 2017). Systematic comparison of the folding of Kt-7 and the box C/D k-turns showed that −1b:−1n = C:G confers folding in metal ions, while inversion to −1b:−1n = G:C prevents folding under the same conditions.…”

“…These rules appear to apply generally for all k-turns studied. Importantly, every k-turn that has been analyzed, irrespective of the 3b:3n or −1b:−1n sequences, undergoes folding into the kinked conformation on addition of L7Ae protein (McPhee et al 2014;Ashraf et al 2017).…”

“…Importantly, even those k-turns that do not fold on addition of metal ions generally fold on binding L7Ae (McPhee et al 2014) or 15.5K (Huang et al 2017) proteins. Examples of k-turns that exhibit this behavior are the archaeal box C/D k-turns (Ashraf et al 2017) and the human U4 snRNA k-turn (McPhee et al 2014).…”