Folding energy landscape of the thiamine pyrophosphate riboswitch aptamer.

Riboswitches are motifs in the untranslated regions (UTRs) of RNA transcripts that sense metabolite levels and modulate the expression of the corresponding genes for metabolite import, export, synthesis, or degradation. All riboswitches contain an aptamer: an RNA structure that, upon binding ligand, folds to expose or sequester regulatory elements in the adjacent sequence through alternative nucleotide pairing. The coupling between ligand binding and aptamer folding is central to the regulatory mechanisms of thiamine pyrophosphate (TPP) riboswitches and has not been fully characterized. Here, we show that TPP aptamer folding can be decomposed into ligand-independent and -dependent steps that correspond to the formation of secondary and tertiary structures, respectively. We reconstructed the full energy landscape for folding of the wild-type (WT) aptamer and measured perturbations of this landscape arising from mutations or ligand binding. We show that TPP binding proceeds in two steps, from a weakly to a strongly bound state. Our data imply a hierarchical folding sequence, and provide a framework for understanding molecular mechanism throughout the TPP riboswitch family.

Electrostatic interactions in a peptide--RNA complex.

Parallel experimental measurements and theoretical calculations have been used to investigate the energetics of electrostatic interactions in the complex formed between a 22 residue, alpha-helical peptide from the N protein of phage lambda and its cognate 19 nucleotide box B RNA hairpin. Salt-dependent free energies were measured for both peptide folding from coil to helix and peptide binding to RNA, and from these the salt-dependence of binding pre-folded, helical peptide to RNA was determined ( partial differential (DeltaG degrees (dock))/ partial differential log[KCl]=5.98(+/-0.21)kcal/mol). (A folding transition taking place in the RNA hairpin loop was shown to have a negligible dependence on salt concentration.) The non-linear Poisson-Boltzmann equation was used to calculate the same salt dependence of the binding free energy as 5.87(+/-0.22)kcal/mol, in excellent agreement with the measured value. Close agreement between experimental measurements and calculations was also obtained for two variant peptides in which either a basic or acidic residue was replaced with an uncharged residue, and for an RNA variant with a deletion of a single loop nucleotide. The calculations suggest that the strength of electrostatic interactions between a peptide residue and RNA varies considerably with environment, but that all 12 positive and negative N peptide charges contribute significantly to the electrostatic free energy of RNA binding, even at distances up to 11A from backbone phosphate groups. Calculations also show that the net release of ions that accompanies complex formation originates from rearrangements of both peptide and RNA ion atmospheres, and includes accumulation of ions in some regions of the complex as well as displacement of cations and anions from the ion atmospheres of the RNA and peptide, respectively.

RNA BIOCHEMISTRY. Factor-dependent processivity in human eIF4A DEAD-box helicase.

During eukaryotic translation initiation, the small ribosomal subunit, assisted by initiation factors, locates the messenger RNA start codon by scanning from the 5' cap. This process is powered by the eukaryotic initiation factor 4A (eIF4A), a DEAD-box helicase. eIF4A has been thought to unwind structures formed in the untranslated 5' region via a nonprocessive mechanism. Using a single-molecule assay, we found that eIF4A functions instead as an adenosine triphosphate-dependent processive helicase when complexed with two accessory proteins, eIF4G and eIF4B. Translocation occurred in discrete steps of 11 ± 2 base pairs, irrespective of the accessory factor combination. Our findings support a memory-less stepwise mechanism for translation initiation and suggest that similar factor-dependent processivity may be shared by other members of the DEAD-box helicase family.

Folding and unfolding single RNA molecules under tension.

Single-molecule force spectroscopy constitutes a powerful method for probing RNA folding: It allows the kinetic, energetic, and structural properties of intermediate and transition states to be determined quantitatively, yielding new insights into folding pathways and energy landscapes. Recent advances in experimental and theoretical methods, including fluctuation theorems, kinetic theories, novel force clamps, and ultrastable instruments, have opened new avenues for study. These tools have been used to probe folding in simple model systems, for example, RNA and DNA hairpins. Knowledge gained from such systems is helping to build our understanding of more complex RNA structures composed of multiple elements, as well as how nucleic acids interact with proteins involved in key cellular activities, such as transcription and translation.