Programmed −1 frameshifting, whereby the reading frame of a ribosome on messenger RNA is shifted in order to generate an alternate gene product, is often triggered by a pseudoknot structure in the mRNA in combination with an upstream slippery sequence. The efficiency of frameshifting varies widely for different sites, but the factors that determine frameshifting efficiency are not yet fully understood. Previous work has suggested that frameshifting efficiency is related to the resistance of the pseudoknot against mechanical unfolding. We tested this hypothesis by studying the mechanical properties of a panel of pseudoknots with frameshifting efficiencies ranging from 2% to 30%: four pseudoknots from retroviruses, two from luteoviruses, one from a coronavirus, and a nonframeshifting bacteriophage pseudoknot. Using optical tweezers to apply tension across the RNA, we measured the distribution of forces required to unfold each pseudoknot. We found that neither the average unfolding force, nor the unfolding kinetics, nor the parameters describing the energy landscape for mechanical unfolding of the pseudoknot (energy barrier height and distance to the transition state) could be correlated to frameshifting efficiency. These results indicate that the resistance of pseudoknots to mechanical unfolding is not a primary determinant of frameshifting efficiency. However, increased frameshifting efficiency was correlated with an increased tendency to form alternate, incompletely folded structures, suggesting a more complex picture of the role of the pseudoknot involving the conformational dynamics.force spectroscopy | gene regulation | RNA folding | translation
The duration of structural transitions in biopolymers is only a fraction of the time spent searching diffusively over the configurational energy landscape. We found the transition time, τ(TP), and the diffusion constant, D, for DNA and RNA folding using energy landscapes obtained from single-molecule trajectories under tension in optical traps. DNA hairpins, RNA pseudoknots, and a riboswitch all had τ(TP)~10 μs and D~10(-13-14) m(2)/s, despite widely differing unfolding rates. These results show how energy-landscape analysis can be harnessed to characterize brief but critical events during folding reactions.
The precise excision of introns from precursor mRNAs (pre-mRNAs) in eukaryotes is accomplished by the spliceosome, a complex assembly containing five small nuclear ribonucleoprotein (snRNP) particles. Human p14, a component of the spliceosomal U2 and U11͞U12 snRNPs, has been shown to associate directly with the pre-mRNA branch adenosine early in spliceosome assembly and within the fully assembled spliceosome. Here we report the 2.5-Å crystal structure of a complex containing p14 and a peptide derived from the p14-associated U2 snRNP component SF3b155. p14 contains an RNA recognition motif (RRM), the surface of which is largely occluded by a C-terminal ␣-helix and a portion of the SF3b155 peptide. An analysis of RNA⅐protein crosslinking to wildtype and mutant p14 shows that the branch adenosine directly interacts with a conserved aromatic within a pocket on the surface of the complex. This result, combined with a comparison of the structure with known RRMs and pseudoRRMs as well as modelbuilding by using the electron cryomicroscopy structure of a spliceosomal U11͞U12 di-snRNP, suggests that p14⅐SF3b155 presents a noncanonical surface for RNA recognition at the heart of the mammalian spliceosome.RNA-binding proteins ͉ RNA splicing ͉ spliceosome P recursor mRNA (pre-mRNA) splicing occurs through two sequential transesterification reactions. The first step involves displacement of the 5Ј exon by the nucleophilic attack of the 2Ј hydroxyl of the conserved branch region adenosine. In the second step, the free 5Ј exon attacks the 3Ј splice site to yield the ligated mRNA product. Assembly of the spliceosomal catalytic machinery containing the U1, U2, and U4͞U5͞U6 small nuclear ribonucleoproteins (snRNPs) is directed by conserved sequence elements at the splice sites and within the intron including the branch region (1-3). Spliceosome assembly occurs in an ordered, stepwise fashion through a series of discrete intermediates and involves sequential steps of pre-mRNA recognition by protein and snRNA components of the spliceosome as well as formation and rearrangement of snRNA⅐snRNA interactions. The fully assembled spliceosome contains a U2͞U6 snRNA structure that has been proposed to form the active site for catalysis of the transesterifications (4, 5). Although pre-mRNA splicing is therefore believed to be intrinsically RNA catalyzed, RNA⅐protein crosslinking studies have established direct association of human p14 with the reactive branch adenosine of the pre-mRNA (6, 7). Thus, the catalytic heart of the spliceosome may include protein as well as RNA components.Initial recognition of the pre-mRNA includes a stable U1 snRNP⅐5Ј splice-site base pairing and the association of nonsnRNP protein factors with the branch region͞3Ј splice site to form the early or commitment complex. The ATP-dependent transition to A complex involves the stable association of U2 snRNP with the pre-mRNA, an interaction that includes the formation of a duplex between U2 snRNA and the pre-mRNA branch region. The bulging of the branch adenosine from...
Programmed -1 ribosomal frameshifting (-1 PRF) stimulated by mRNA pseudoknots regulates gene expression in many viruses, making pseudoknots potential targets for anti-viral drugs. The mechanism by which pseudoknots trigger -1 PRF, however, remains controversial, with several competing models. Recent work showed that high -1 PRF efficiency was linked to high pseudoknot conformational plasticity via the formation of alternate conformers. We tested whether pseudoknots bound with an anti-frameshifting ligand exhibited a similar correlation between conformational plasticity and -1 PRF efficiency by measuring the effects of a ligand that was found to inhibit -1 PRF in the SARS coronavirus on the conformational dynamics of the SARS pseudoknot. Using single-molecule force spectroscopy to unfold pseudoknots mechanically, we found that the ligand binding effectively abolished the formation of alternate conformers. This result extends the connection between -1 PRF and conformational dynamics and, moreover, suggests that targeting the conformational dynamics of pseudoknots may be an effective strategy for anti-viral drug design.
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