Functional complexity and regulation through RNA dynamics

Abstract

Changes to the conformation of coding and non-coding RNAs form the basis of elements of genetic regulation and provide an important source of complexity, which drives many of the fundamental processes of life. Although the structure of RNA is highly flexible, the underlying dynamics of RNA are robust and are limited to transitions between the few conformations that preserve favourable base-pairing and stacking interactions. The mechanisms by which cellular processes harness the intrinsic dynamic behaviour of RNA and use it within functionally productive pathways are complex. The versatile functions and ease by which it is integrated into a wide variety of genetic circuits and biochemical pathways suggests there is a general and fundamental role for RNA dynamics in cellular processes.

Figure 1

Shape and form of RNA dynamics. (A) A RNA free energy landscape (green). Shown are secondary and tertiary RNA conformations of low-lying energy. The relative population of each conformation is indicated using red balls. Cellular effectors (bolts) can modify the energy landscape to favor an alternative secondary structure (top), or preferentially stabilize an alternate tertiary conformation (bottom). (B) Exchange between alternative, isoenergetic secondary structures that are separated by large energetic barriers due to disruption of base-pairs in the transition state. Note that RNA helices tend to be shorter than ~15 base pairs. (C) The accessible range of inter-helical conformations for an RNA two-way junction consisting of a trinucleotide bulge, with the possible paths of the bulge, which were excluded during the modeling, illustrated as cartoons (red)^,. The allowed range of conformations is restricted towards a specific and directed conformational pathway by steric and stereochemical forces. (D) Flipping out of a non-canonical base pair with an RNA internal loop (red) from an intra-helical stacked to extra-helical unstacked conformation. The motion occurs without perturbing flanking Watson-Crick pairs (green).

Figure 2

Triggering RNA conformational transitions. (A) Conformational changes in the spliceosomal U4 snRNA K-turn motif (2KR8) triggered upon binding to the protein hPrp31-15.5K (2OZB). (B) Similarity between the TAR RNA inter-helical conformational conformations that are triggered by binding to small molecules (in grey) and that are sampled by equilibrium dynamics (in green) in the unbound state. Adapted from. (C) RNA conformational transitions during spliceosome assembly on pre-mRNA (dashed line). (D) Modulating RNA structure by steering the co-transcriptional folding pathway, with the adenine transcription terminating riboswitch as a prototypical example. Shown is the progression of co-transcriptional folding with and without the ligand. The RNA polymerase is shown in gold. (E) Examples of tandem riboswitch architectures. Cooperative binding of glycine by the gly riboswtich using tandem aptamer domains and one expression platform (left panel). Tandem SAM and AdoCbl riboswitches in which either of two ligands yields an output of gene repression (right panel). Transcription terminator stems are shown in red. (F) Conformations of HDV ribozyme pre-cleavage with Mg²⁺ (1VC7) and post-cleavage (1DRZ). The catalytic core is highlighted in the two states, with the substrate and Mg²⁺ ion shown in green and yellow, respectively. (G) Melting of secondary structure around the ribosome binding site of virulence genes in the pathogen triggered by an increase in temperature makes the Shine-Dalgarno sequence available for ribosome binding and translation initiation.

Figure 3

Functional outputs of secondary structural changes. (A) Transcriptional activation of the aminoacyl-tRNA synthetase gene by uncharged tRNA by steering co-transcriptional folding away from a transcription terminating helix. (B) Translation control of VEGFA expression via a dual protein-dependent RNA secondary structural switch that responds to IFN-γ (left panel) and hypoxic stress (right panel). (C) TPP-riboswitch-regulated alternative splicing and gene expression of NMT1. On binding of TPP, the aptamer domain undergoes a conformational change, which exposes a proximal splice site (diamond). Spliced mRNAs now contain uORFs, thus reducing expression of the NMT1 ORF. (D) Pumilio protein-mediated mRNA secondary structural switch controls accessibility of microRNA binding sites and regulates expression of p27 protein. Binding of PUM1 induces a conformational change to expose the miR-211/miR-222 binding site to allow for p27 silencing. (E) Secondary structural switch couples dimerization and diploid genome packaging of the Moloney murine leukemia virus. Dimerization leads to a coupled frame shift that exposes NC protein binding sites (green) required for genome packaging.

Figure 4

Functional outputs of tertiary conformational changes. (A) X-ray structures of tRNA^Phe in the unbound state (black, 1EHZ), in complex with RNaseP (blue, engineered anticodon stem removed, 3Q1Q), the ribosome in the P/E state (green, 3R8N), isopentenyl-tRNA transferase (red, 3FOZ), and phenyalanyl-tRNA synthetase (yellow, 1EIY). The structures are superimposed by the acceptor stem. (B) Hierarchical assembly of the central domain of the 30S ribosomal subunit by successive protein-induced changes in the conformation of 16S rRNA. (C) Enzymatic cycle of the hairpin ribozyme. (D) Ratcheting motions of the ribosome as observed by X-ray crystallography. The degree of 30S subunit atomic displacement between the unratcheted and R₂ ratcheted states with the 50S subunit as a reference (not shown) are color-coded by Å (left). Atomic displacement vectors and arrows indicate the directionality of the change (right). From reference. Reprinted with permission from AAAS. (E) Free energy landscape of ribosomal ratcheting, as calculated from sub-classification of cryo-EM particles. Movements of the 30S subunit body and head domains in relation to the 50S subunit are shown in units of degrees and arbitrary units, respectively, with corresponding tRNA translocation intermediates outlined in black. Reprinted by permission from Macmillan Publishers Ltd: Nature, copyright (2009). (F) Dynamics of the 50S ribosomal L1 stalk monitored by single molecule FRET. Representative smFRET trace (top) and histogram (bottom left) of the L1 stalk dynamically sampling open and closed conformations in A and P-site tRNA-bound ribosome complexes. Upon translocation by EF-G • GTP and tRNA occupation of the E and P-sites the L1 stalk conformation shifts dramatically (bottom right). Adapted with permission from the National Academy of Sciences, USA.