Evaluating RNA Structural Flexibility: Viruses Lead the Way

Abstract

Our understanding of RNA structure has lagged behind that of proteins and most other biological polymers, largely because of its ability to adopt multiple, and often very different, functional conformations within a single molecule. Flexibility and multifunctionality appear to be its hallmarks. Conventional biochemical and biophysical techniques all have limitations in solving RNA structure and to address this in recent years we have seen the emergence of a wide diversity of techniques applied to RNA structural analysis and an accompanying appreciation of its ubiquity and versatility. Viral RNA is a particularly productive area to study in that this economy of function within a single molecule admirably suits the minimalist lifestyle of viruses. Here, we review the major techniques that are being used to elucidate RNA conformational flexibility and exemplify how the structure and function are, as in all biology, tightly linked.

Keywords: NMR; RNA flexibility; RNA structure; RNA viruses; SAXS; SHAPE; proximity ligation; smFRET.

Figure 1

The HIV-1 genomic RNA leader contains a structural switch. Left hand panel: pseudoknot structure, where the DIS palindrome forms a pseudoknot with upstream U5 sequences. Right hand panel: the same U5 sequence pairs with the Gag start codon to form the U5:AUG helix. Structures that change during the switch are labelled in red. Structures are drawn according to data from Kenyon et al., 2013.

Figure 2

Process diagram for high-throughput SHAPE structure determination. The 1M7 reacts covalently with a 2′OH on the sample RNA. This occurs preferentially where the backbone is flexible. Reverse transcriptase generates a family of cDNAs of length corresponding to the sites of 1M7 adduct formation. Comparison with a sequencing ladder allows readout of amount of cDNA product of each length. This ‘SHAPE reactivity’ is used to inform minimal free energy prediction software.

Figure 3

Process diagram for proximity ligation. Although the specifics of different proximity ligation methods vary, many follow the general scheme shown above. The modular nature of the scheme allows a mixing and matching of different steps to suit the experimental goals.

Figure 4

Principle of single molecule FRET (sm-FRET). sm-FRET relies upon the spatial proximity of a donor and acceptor dye. Where the dyes are far apart, there is little resonance energy transfer and a low E value; conversely, where the dyes are much closer to each other, transfer efficiency is greatly increased. Emission from the acceptor dye is a measure of proximity.

Figure 5

Structure of the HCV IRES. The two major domains, II and III, are highlighted, alongside the pseudoknot.