RNA Folding and Unfolding Under Force: Single-Molecule Experiments and Their Analysis.

We have previously described (Geffroy et al. Methods Mol Biol 1665:25-40, 2018) how to unfold (or fold) a single RNA molecule under force using a dual-beam optical trap setup. In this chapter, we complementarily describe how to analyze the corresponding data and how to interpret it in terms of RNA three-dimensional structure.

Overstretching Double-Stranded RNA, Double-Stranded DNA, and RNA-DNA Duplexes.

Using single-molecule force measurements, we compare the overstretching transition of the four types of duplexes composed of DNA or RNA strands. Three of the four extremities of each double helix are attached to two microscopic beads, and a stretching force is applied with a dual-beam optical trapping interferometer. We find that overstretching occurs for all four duplexes with small differences between the plateau forces.

Force measurements show that uL4 and uL24 mechanically stabilize a fragment of 23S rRNA essential for ribosome assembly.

In vitro reconstitution studies have shown that ribosome assembly is highly cooperative and starts with the binding of a few ribosomal (r-) proteins to rRNA. It is unknown how these early binders act. Focusing on the initial stage of the assembly of the large subunit of the ribosome, we prepared a 79-nucleotide-long region of 23S rRNA encompassing the binding sites of the early binders uL4 and uL24.

Single-Molecule FRET Assay to Observe the Activity of Proteins Involved in RNA/RNA Annealing.

In recent years, single-molecule fluorescence resonance energy transfer (smFRET) has emerged as a powerful technique to study macromolecular interactions. The chief advantages of smFRET analysis compared to bulk measurements include the possibility to detect sample heterogeneities within a large population of molecules and the facility to measure kinetics without needing the synchronization of intermediate states. As such, the methodology is particularly well adapted to observe and analyze RNA/RNA and RNA/protein interactions involved in small noncoding RNA-mediated gene regulation networks.

RNA Unzipping and Force Measurements with a Dual Optical Trap.

In order to mechanically unfold a single RNA molecule, an RNA/DNA hybrid construction is prepared which allows specific attachment to two micrometer-sized beads. A dual-beam optical trap thus holding the construct in solution captures the beads separately. Unfolding of a molecule is obtained by increasing the distance between the traps, one trap being slowly moved while the other is held fixed.

A FRET-based, continuous assay for the helicase activity of DEAD-box proteins.

Enzymes from cold-adapted organisms are generally endowed with lower activation enthalpies than their counterparts from organisms growing at higher temperatures, making them better catalysts in the cold. However, the enzymes of RNA metabolism have not been examined in this respect. A challenge for studying cold adaptation of DEAD-box RNA helicases is the low precision of the classical, discontinuous helicase assay based on electrophoretic separation of duplexes and isolated strands.

Functions of DEAD-box proteins in bacteria: current knowledge and pending questions.

DEAD-box proteins are RNA-dependent ATPases that are widespread in all three kingdoms of life. They are thought to rearrange the structures of RNA or ribonucleoprotein complexes but their exact mechanism of action is rarely known. Whereas in yeast most DEAD-box proteins are essential, no example of an essential bacterial DEAD-box protein has been reported so far; at most, their absence results in cold-sensitive growth.

Probing ribosomal protein-RNA interactions with an external force.

Ribosomal (r-) RNA adopts a well-defined structure within the ribosome, but the role of r-proteins in stabilizing this structure is poorly understood. To address this issue, we use optical tweezers to unfold RNA fragments in the presence or absence of r-proteins. Here, we focus on Escherichia coli r-protein L20, whose globular C-terminal domain (L20C) recognizes an irregular stem in domain II of 23S rRNA.

Cold adaptation in DEAD-box proteins.

Spontaneous rearrangements of RNA structures are usually characterized by large activation energies and thus become very slow at low temperatures, yet RNA structure must remain dynamic even in cold-adapted (psychrophilic) organisms. DEAD-box proteins constitute a ubiquitous family of RNA-dependent ATPases that can often unwind short RNA duplexes in vitro (helicase activity), hence the belief that one of their major (though not exclusive) roles in vivo is to assist in RNA rearrangements. Here, we compare two Escherichia coli DEAD-box proteins and their orthologs from the psychrophilic bacteria Pseudoalteromonas haloplanktis and Colwellia psychrerythraea from the point of view of enzymatic properties.

Quaternary structure and biochemical properties of mycobacterial RNase E/G.

The RNase E/G family of endoribonucleases plays the central role in numerous post-transcriptional mechanisms in Escherichia coli and, presumably, in other bacteria, including human pathogens. To learn more about specific properties of RNase E/G homologues from pathogenic Gram-positive bacteria, a polypeptide comprising the catalytic domain of Mycobacterium tuberculosis RNase E/G (MycRne) was purified and characterized in vitro. In the present study, we show that affinity-purified MycRne has a propensity to form dimers and tetramers in solution and possesses an endoribonucleolytic activity, which is dependent on the 5'-phosphorylation status of RNA.

Characterization of Aquifex aeolicus RNase E/G.

The RNase E/G homologue from the thermophilic eubacterium Aquifex aeolicus has been overexpressed in Escherichia coli, purified, and characterized in vitro. We show that A. aeolicus RNase E/G has a temperature-dependent, endoribonucleolytic activity.

Studies on three E. coli DEAD-box helicases point to an unwinding mechanism different from that of model DNA helicases.

DEAD-box proteins participate in various aspects of RNA metabolism in all organisms. These RNA-dependent ATPases are usually regarded as double-stranded RNA unwinding enzymes, though in vitro this activity has only been demonstrated for a subset of them. Given their high biological specificity, their equivocal unwinding activity may reflect the noncognate character of the substrates used in vitro.

Structure of a circularly permuted phosphoglycerate kinase.

The crystallographic structure of a circularly permuted form of yeast PGK, 72p yPGK, has been determined to a resolution of 2.3 A by molecular replacement. In this engineered protein, the C- and N-terminal residues of the wild-type protein are directly connected by a peptide bond and new N- and C-terminal residues are located within the N-terminal domain.