Biophysics of Infection by Mark C. Leake

Author:Mark C. Leake
Language: eng
Format: epub
Publisher: Springer International Publishing, Cham

11.6.3.2 Single-Molecule Force Studies on the Adenine Aptamer

The application of single-molecule optical force to investigate the folding landscape of a riboswitch was pioneered by S. Block and co-workers using the pbuE aptamer from B. subtillis (Greenleaf et al. 2008) under constant-force and force-ramp regimes. In their initial configuration, they used a dual-beam optical trap to monitor the conformational landscape of an add riboswitch being transcribed in situ. The RNA polymerase from E. coli was attached to one of the beads and transcriptionally stalled downstream of promoter site. Whilst stalled, the initial 29 nt transcript emerging from the RNAP was hybridized to a DNA handle attached to the other bead, thus providing a dual-beam ‘dumbbell’ geometry to monitor FEC between the RNAP and the transcribed RNA once the transcription was restarted by introducing nucleoside triphosphates. To avoid disassembly of the complex once the aptamer sequence was transcribed, a road block consisting of a streptavidin molecule bound to a biotin group placed at the 5′ termini of the DNA template.

Using a combination of constant-force and force-ramp methods, a transcriptional landscape consisting of five distinct states was observed. In addition to demonstrating that the formation of the P2 and P3 loops takes place before the assembly of the ligand-binding pocket, they uncovered a ligand-binding intermediate state, termed A-comp, formed prior to the native fully folded state. A later study of the add aptamer from V. vulnificus (Neupane et al. 2011) also revealed a similar five-state free energy folding landscape and an intermediate conformation, termed P1_U, located before the fully folded state and after the formation of the P2 and P3 stems. It was suggested that this P1_U state may arise from the docking of the P2 and P3 stem loops and the RNA junction partially organized but with the P1 stem still unfolded. To date, it remains to be determined whether the intermediate state (I) observed by smFRET (Lemay et al. 2006) and this P1_U state observed by single-molecule force are somehow structurally related. The single-molecule free energy folding trajectory obtained for both aptamers suggests that weak tertiary interactions are a key to promote the long-range conformational changes needed to close the P1 switching stem. Thus, both aptamers can be added to the growing number of examples challenging the generality of a strictly hierarchical RNA folding model, where secondary structures are typically formed first and followed by the formation of tertiary contacts (Cruz and Westhof 2009).

It is interesting to note that these single-molecule force experiments detected significant differences for the transition from a pre-organized P1_U (add aptamer) or A-comp (pbuE aptamer) to the fully folded native state. These differences were not only on the degree of stabilization of the fully folded state provided by ligand-binding, which was 4 kcal mol−1 for the pbuE aptamer compared to 8 kcal mol−1 for the add, but also on the nature of key interactions involved in transition from P1_U or A-comp to the ligand-bound state, whereas for the pbuE aptamer, a GC base pair at the base

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