Journal of Molecular Biology
Single-Molecule Fluorescence Resonance Energy Transfer Assays Reveal Heterogeneous Folding Ensembles in a Simple RNA Stem–Loop
Introduction
Single-stranded, positive-sense RNA viruses are the most common species in the virosphere.1 They all face a common challenge of packaging their nucleic acids, which also need to serve as mRNAs and replication templates, each with its own requirement to adopt extended unstructured conformations into a protective shell of protein subunits. The stratagem that many single-stranded RNA viruses have evolved is to assemble their capsids around their genomes. A major unresolved problem in structural virology, however, is to understand the detailed molecular mechanisms that give rise to the assembly of capsids of the correct size and symmetry. This is especially true for viruses whose capsids exhibit quasi-equivalent symmetry in their coat protein (CP) lattices.2 Various proposals for formation of protein shells based on the initial formation of three-fold3 or five-fold4, 5 assembly initiation complexes have been made, but there is as yet no complete molecular description of an entire assembly pathway.
The RNA bacteriophage MS2 is an ideal model for investigating such phenomena owing to the extensive biochemical and structural data that are available.6, 7, 8, 9 It has a single-stranded, positive-sense RNA genome of 3569 nucleotides that encodes only four gene products: CP, replicase protein, lysis protein and maturation protein. CP is the most highly expressed of these four gene products, and 180 copies, in the form of 90 interdigitated non-covalent dimers (CP2), assemble to form a T = 3 icosahedral protein shell that encapsidates the genome in the mature virion. The levels of gene expression are known to be regulated significantly by a series of ordered RNA folding/unfolding events.10 Capsid reassembly in vitro, and presumably assembly in vivo, can be triggered by a sequence-specific RNA–protein interaction between a CP dimer and an RNA stem–loop (TR; see Fig. 1) of only 19 nucleotides located between positions 1746 and 1764 within the genomic sequence. The complex has provided a wealth of structural data on the basis of the sequence-specific recognition events.7, 9, 11 The TR sequence encompasses the start codon of the viral replicase, and CP dimer binding leads to translational repression of this cistron. Complex formation also results in an allosteric conformational change within the protein, taking it from a largely symmetric structure to an asymmetric one. Using both mass spectrometry and size-exclusion chromatography, we have shown previously that the TR:CP2 complex is kinetically trapped and forms capsids only very slowly. In contrast, when this complex is added to excess RNA-free protein, T = 3 shells form rapidly, implying that the differing dimer conformers present in this mixture correspond to the known quasi-equivalent CP dimer conformations seen in X-ray structures of the T = 3 shell (i.e., A/B- and C/C-like species).12
This represents one of the most detailed molecular models for the switching of quasi-equivalent conformations available for any viral system. Allosteric, TR RNA-induced conformational switching in reassembly assays, however, raises the question of how similar effects are achieved during packaging of genomic RNA. A recent cryo-electron microscopic medium-resolution reconstruction of the wild-type phage13 revealed a unique ordering of almost the entire genomic RNA into a double-shell structure. One of these shells of RNA density is closely associated with the CP dimers within the protein capsid. This averaged RNA density occupies the approximate position of the TR stem–loop with respect to each dimer, but the averaged density below A/B and C/C dimers is different. This result raises the possibility that nucleotide sequences other than TR mimic its allosteric effect at appropriate spatial positions during capsid assembly.
It is therefore important to understand the kinetics of folding/unfolding of the TR stem–loop, which creates the initial target for CP binding, the first step in the assembly pathway. NMR, optical spectroscopy and biochemical structure probing all confirm that RNA fragments encompassing TR exist as stem–loops able to adopt a range of distinct conformers.14, 15, 16 CP dimer–TR RNA complexes contain only one of these conformers (Fig. 1), selecting what appears to be a minor conformer from within the ensemble of solution structures.7, 9 In order to probe the details of the stem–loop folding reaction, we used single-molecule fluorescence resonance energy transfer (SM-FRET) and fluorescence correlation spectroscopy (FCS). Thermal unfolding of RNA stem–loops derivatised with appropriate donor and acceptor dyes on the 5′ and 3′ ends, respectively, has been used to examine stability. In addition to the wild-type sequence, we studied a number of sequence variants in order to be able to relate sequence/structure to folding behaviour. The results show that the wild-type RNA samples both folded (stem–loop) and unfolded conformational ensembles are separated by a free energy barrier. Unfolding appears to be nucleated by single-stranded interruptions to base-paired segments. Strikingly, a single-nucleotide substitution in the loop (C-5 variant) has dramatic effects on the (un)folding behaviour, creating RNA population ensembles that are more heterogeneous. The biological implications of this behaviour for the phage are discussed.
Section snippets
Preparation and characterisation of TR oligonucleotides
Three oligonucleotides encompassing the TR stem–loop (Fig. 1) and some sequence variants were synthesised by solid-phase techniques starting with a 3′-fluorescein controlled pore glass derivative† and terminating with a 5′-amino linker. Details of the synthesis, deprotection and purification were as described previously,17 and the final products were characterised by mass spectrometry. The variants were chosen to probe how the detailed sequence and secondary structure of
Discussion
The importance of RNA biology28, 29 and its potential applications in disease therapy have highlighted the need for better understanding of the folding pathways for this important class of molecules. This has led to a number of SM studies mostly directed at understanding the molecular mechanism of RNA catalysis and the folding of small ribozymes into functional states.30, 31, 32 RNA stem–loop formation has previously been probed by temperature-jump experiments, and, more recently, the folding
Chemical synthesis of RNAs
Synthesis, deprotection and purification of RNA oligonucleotides were carried out as described previously17 with the following modifications: 3′-(6-fluorescein) controlled pore glass beads (Glen Research) were used to produce oligonucleotides derivatised with a donor fluorophore. A 5′-TFA (trifluoroacetyl) amino modifier was incorporated at the 5′ ends to allow subsequent addition of the acceptor dye.43 The sequences of the three RNA stem–loops are shown below:
Wild-type MS2 TR: 5′-ACA UGA GGA
Acknowledgements
We thank Drs. Chris Adams for providing help in the initial RNA synthesis, purification and derivatisation; Alison Ashcroft for mass spectrometry measurements; and Claire Friel, Roman Tuma and David Millar (The Scripps Research Institute, La Jolla) for useful discussions and comments on the manuscript. We also thank Dr. Máire Convery for helping with the structural figure. P.G.S. thanks the UK Medical Research Council and the Leverhulme Trust for financial support; T.S. and J.J.W. were
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Present addresses: C. Gell, Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, 01307 Dresden, Germany; T. Sabir, ParticlesCIC, Houldsworth Building, University of Leeds, Leeds LS2 9JT, UK; A. Rashid, The John Innes Centre, Colney Lane, Norwich NR4 7UH, UK; D. A. M. Smith, Avacta Group PLC, York Biocentre, York Science Park, Innovation Way, Heslington, York YO10 5NY, UK.