Journal of Molecular Biology
Two Distinct Binding Modes of a Protein Cofactor with its Target RNA
Introduction
Ribonucleoprotein (RNP) complexes catalyze many essential cellular reactions, including important examples such as protein synthesis and messenger RNA processing. In these RNPs, the catalytic site is often formed by the RNA while the protein cofactors play auxiliary roles.1 These enzymatic RNAs must fold to specific structures to function properly. In vitro, RNA folding tends to be difficult for two reasons: the energy landscape of the RNA is rugged with kinetic traps that prevent efficient folding or the active state of the RNA is only marginally stable.2., 3., 4., 5., 6., 7., 8., 9., 10., 11., 12., 13., 14., 15. In cells, these difficulties are mitigated in part by the association of RNA with protein cofactors that facilitate RNA folding.12., 13., 14., 15.
Proteins appear to facilitate the folding of RNA via two broad mechanisms.12,13 In the first mechanism, proteins interact non-specifically with the RNA and promote RNA folding by resolving non-native conformations, in a way analogous to chaperones acting on misfolded proteins.13,16,17 The non-specific nature of these RNA–protein interactions may also inhibit RNA folding under certain conditions, by disrupting native RNA structures as well as misfolded ones. In a second mechanism, a specific protein cofactor binds to well-defined structural features of its target RNA, stabilizing the native RNA structure. If the protein cofactor binds to the RNA at an early step in the folding pathway, the protein may effectively nucleate subsequent RNA folding.11,18 Alternatively, the protein cofactor may capture and stabilize a transiently formed native RNA structure, rather than actively inducing structural changes in the RNA, a mechanism referred to as tertiary structure capture. This has been thought to be the mechanism by which CBP2 facilitates the folding of the yeast mitochondrial bI5 group I intron.19,20 A protein cofactor may also facilitate RNA folding by a mechanism that exhibits features of both tertiary structure nucleation or capture.21,22 An RNA molecule could also rely on both a chaperone and a distinct specific cofactor working in concert to accomplish efficient folding.23 The level of complexity observed for protein-facilitated RNA folding makes the acquisition of real-time information on folding dynamics critical for a comprehensive understanding of folding mechanisms.
In this work, we use fluorescence resonance energy transfer (FRET)24,25 to investigate the real-time folding dynamics of RNP complexes at the single-molecule level. It has been shown that single-molecule FRET can be used to detect conformational changes in a small RNA three-helix junction induced by a specific ribosomal protein and in a DNA stem-loop induced by an RNA chaperone.26,27 A useful property of the single-molecule approach is its ability to detect non-accumulative folding intermediates and multiple folding pathways that are potentially difficult to detect in ensemble measurements.6,28., 29., 30., 31. Here, we report a dynamic structure for the bI5 group I intron, and two binding modes between this RNA and its protein cofactor, CBP2. While the specific CBP2-RNA binding mode stabilizes native RNA structures, the non-specific binding mode of CBP2 causes large conformational fluctuations in the RNA. Before attaining its native structure, the bI5 RNA folds through fluctuating intermediate states induced by non-specific CBP2 binding. These results suggest a complex assembly mechanism that involves both non-specific and specific interactions by a protein cofactor with its RNA target, which ultimately lead to formation of a well-defined and active state.
Section snippets
Preparation of bI5 for investigation by single-molecule FRET
The bI5 RNP is comprised of the bI5 group I intron RNA and its CBP2 protein cofactor. The bI5 RNA exhibits splicing activity while CBP2 facilitates folding of the RNA.10,32., 33., 34. The bI5 RNA contains three major domains: the P5-P4-P6 and P7-P3-P8 domains, which constitute the conserved catalytic core of all group I introns, and the 5′ domain, which spans helices P1-P2-P2a (Figure 1(a)).
To probe the folding of bI5 molecules with FRET, we attached FRET donor (Cy3) and acceptor (Cy5) dyes
Discussion
Nearly all cellular RNA enzymes rely on the help of proteins that bind either specifically or non-specifically in order to function properly. In many cases, the role of these proteins is to help the catalytic RNA adopt its native structure, rather than to catalyze the underlying enzymatic reaction. These proteins function in part by altering the conformational dynamics of the RNA. In this work, we have used single-molecule FRET to monitor the conformational dynamics of the bI5 RNA prior to
The bI5 RNA and CBP2 protein preparation
All fluorescently labeled RNA constructs consist of three oligonucleotides: an in vitro transcribed RNA that spans the majority of the bI5 sequence, plus two synthetic RNA or DNA oligonucleotides (Dharmacon and Qiagen Operon, respectively) labeled with Cy5 or Cy3. One of the oligonucleotides is also labeled with biotin to allow surface immobilization. The sequences used for each DNA or RNA oligo are listed in Table 1 of the Supplementary Data. Cy3 or Cy5 were either attached to the oligos
Acknowledgements
This work is supported in part by the Office of Naval Research, the National Science Foundation, a Packard Science and Engineering Fellowship, and the Howard Hughes Medical Institute (to X.Z.); and by the National Institutes of Health (GM 56222 to K.M.W.). G.B. is supported in part by a NIH training grant in Molecular, Cellular and Chemical Biology. L.G.N. is a Fannie and John Hertz pre-doctoral fellow.
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