Two Distinct Binding Modes of a Protein Cofactor with its Target RNA

https://doi.org/10.1016/j.jmb.2006.06.048Get rights and content

Abstract

Like most cellular RNA enzymes, the bI5 group I intron requires binding by a protein cofactor to fold correctly. Here, we use single-molecule approaches to monitor the structural dynamics of the bI5 RNA in real time as it assembles with its CBP2 protein cofactor. These experiments show that CBP2 binds to the target RNA in two distinct modes with apparently opposite effects: a “non-specific” mode that forms rapidly and induces large conformational fluctuations in the RNA, and a “specific” mode that forms slowly and stabilizes the native RNA structure. The bI5 RNA folds though multiple pathways toward the native state, typically traversing dynamic intermediate states induced by non-specific binding of CBP2. These results suggest that the protein cofactor-assisted RNA folding involves sequential non-specific and specific protein–RNA interactions. The non-specific interaction potentially increases the local concentration of CBP2 and the number of conformational states accessible to the RNA, which may promote the formation of specific RNA–protein interactions.

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.

References (42)

  • S. Mohr et al.

    A DEAD-box protein functions as an ATP-dependent RNA chaperone in group I intron splicing

    Cell

    (2002)
  • P.R. Selvin

    Fluorescence resonance energy-transfer

    Methods Enzymol.

    (1995)
  • L.C. Shaw et al.

    Protein-induced folding of a group I intron in cytochrome b pre-mRNA

    J. Biol. Chem.

    (1995)
  • A.E. Webb et al.

    Protein-dependent transition states for ribonucleoprotein assembly

    J. Mol. Biol.

    (2001)
  • H.K. Tirupati et al.

    An RNA binding motif in the Cbp2 protein required for protein-stimulated RNA catalysis

    J. Biol. Chem.

    (1999)
  • R.F. Gesteland et al.

    The RNA world

    (1999)
  • T. Pan et al.

    Intermediates and kinetics traps in the folding of a large ribozyme revealed by circular dichroism and UV absorbance spectroscopies and catalytic activity

    Nature Struct. Biol.

    (1997)
  • D.K. Treiber et al.

    Kinetic intermediates trapped by native interactions in RNA folding

    Science

    (1998)
  • X. Zhuang et al.

    A single molecule study of RNA catalysis and folding

    Science

    (2000)
  • B. Onoa et al.

    Identifying kinetic barriers to mechanical unfolding of the T. thermophila ribozyme

    Science

    (2003)
  • B. Sclavi et al.

    RNA folding at millisecond intervals by synchrotron hydroxyl radical footprinting

    Science

    (1998)
  • Cited by (48)

    • A roadmap for rRNA folding and assembly during transcription

      2021, Trends in Biochemical Sciences
      Citation Excerpt :

      For example, non-native intermediates are thought to contribute to binding of HIV Rev protein to the REV response element (RRE) in the HIV genome, in which binding of multiple Rev proteins promotes stable RNP formation [38]. During cotranscriptional splicing of the yeast mitochondrial b15 group I intron, fast nonspecific binding of the protein Cbp2 hastened the formation of slow, specific RNP interactions that accelerate intron splicing [39]. Finally, it was recently shown that the mouse protein Dazl only resides on a particular mRNA for 1–2 s but nonetheless influences its translation, highlighting that transient RNA-protein binding can trigger changes in gene expression [40].

    • Ensemble and single-molecule biophysical characterization of D17.4 DNA aptamer-IgE interactions

      2016, Biochimica et Biophysica Acta - Proteins and Proteomics
      Citation Excerpt :

      The study of dynamic biological events at the nanoscale has been greatly advanced in recent years by the advent of single molecule spectroscopy (SMS). Recent advances in SMS have made it possible to discern individual dynamical events within complex biological processes such as binding [31–34] and structural fluctuations [35–38]. Single molecule imaging by Total Internal Reflectance Fluorescence (TIRF) microscopy and fluorescence resonance energy transfer are useful in measuring single association/dissociation events without the ensemble averaging that occurs in bulk measurements [39–44].

    • Dynamic Motions of the HIV-1 Frameshift Site RNA

      2015, Biophysical Journal
      Citation Excerpt :

      Flanking basepairs may also have a large effect on RNA dynamics (4,6,20). Despite the enormous size and diversity of RNA transcriptomes (21–23), most of what is known about RNA dynamics has been derived from a relatively small collection of RNAs (19,24–35). Among these, the HIV-1 transactivation response (TAR) RNA element has been studied extensively and has served as a model for investigating RNA motional amplitudes and dynamics (2,4,6,19,20,36–46).

    • The brace for a growing scaffold: Mss116 protein promotes RNA folding by stabilizing an early assembly intermediate

      2012, Journal of Molecular Biology
      Citation Excerpt :

      Other proteins promote RNA folding in the opposite manner, by stabilizing RNA structure.13,14 For example, some proteins function as RNA annealing enzymes that stimulate the formation of RNA secondary structures,18,25,26 while others stabilize transient on-pathway intermediates27–30 or trap the native tertiary structure.31 Multiple studies suggest that ribozymes derived from the yeast mitochondrial group II intron ai5γ fold slowly in vitro as they proceed directly to the native state via several on-pathway intermediate states.3–5

    • Effect of protein binding on RNA folding

      2012, Comprehensive Biophysics
    • Multistep kinetics of the U1A-SL2 RNA complex dissociation

      2011, Journal of Molecular Biology
      Citation Excerpt :

      The contributions of RNA dynamics to complex formation are harder to characterize because the flexibility of the RNA makes structural determination challenging.6,13–19 Investigation of the folding of large RNAs, such as the group I intron, upon binding protein cofactors has revealed long-range allosteric mechanisms that influence binding, folding, and enzyme activity.20,21 The RNA recognition motif (RRM) is one of the most common RNA binding domains and most abundant protein domains in eukaryotes.22

    View all citing articles on Scopus
    View full text