Rajan Lamichhane

RNA looping by PTB: Evidence using FRET and NMR spectroscopy for a role in splicing repression

Alternative splicing plays an important role in generating proteome diversity. The polypyrimidine tract–binding protein (PTB) is a key alternative splicing factor involved in exon repression. It has been proposed that PTB acts by looping out exons flanked by pyrimidine tracts. We present fluorescence, NMR, and in vivo splicing data in support of a role of PTB in inducing RNA loops. We show that the RNA recognition motifs (RRMs) 3 and 4 of PTB can bind two distant pyrimidine tracts and bring their 5′ and 3′ ends in close proximity, thus looping the RNA. Efficient looping requires an intervening sequence of 15 nucleotides or longer between the pyrimidine tracts. RRM3 and RRM4 bind the 5′ and the 3′ pyrimidine tracts, respectively, in a specific directionality and work synergistically for efficient splicing repression in vivo.

Single-molecule FRET of protein-nucleic acid and protein-protein complexes: surface passivation and immobilization.

Single-molecule fluorescence spectroscopy reveals the real time dynamics that occur during biomolecular interactions that would otherwise be hidden by the ensemble average. It also removes the requirement to synchronize reactions, thus providing a very intuitive approach to study kinetics of biological systems. Surface immobilization is commonly used to increase observation times to the minute time scale, but it can be detrimental if the sample interacts non-specifically with the surface. Here, we review detailed protocols to prevent such interactions by passivating the surface or by trapping the molecules inside surface immobilized lipid vesicles. Finally, we discuss recent examples where these methods were applied to study the dynamics of important cellular processes at the single-molecule level.

Exploring RNA folding one molecule at a time.

RNA molecules fold into stable native structures to perform their biological function. RNA folding can be influenced by ions, co-factors, and proteins through numerous mechanisms. Understanding these mechanisms at the molecular level is important for elucidating the structure-function relationship in biologically important RNAs. Recent developments in single molecule spectroscopy have provided new approaches to investigate RNA folding and have allowed identification of kinetic intermediates that would otherwise remain hidden in ensemble-averaged experiments. Here we summarize some of these developments, which provide new insight into the effect of Mg(2+) ions in RNA folding landscapes, the role of cooperativity in RNA tertiary folding, the stepwise folding of RNA during transcription, and the hierarchical assembly of RNA-protein complexes.

Laser-Assisted Single-Molecule Refolding (LASR)

To assemble into functional structures, biopolymers search for global minima through their folding potential energy surfaces to find the native conformation. However, this process can be hindered by the presence of kinetic traps. Here, we present a new single-molecule technique, termed laser-assisted single-molecule refolding (LASR), to characterize kinetic traps at the single-molecule level. LASR combines temperature-jump kinetics and single-molecule spectroscopy. We demonstrate the use of LASR to measure single-molecule DNA melting curves with ∼1°C accuracy and to determine the activation barrier of a model kinetic trap. We also show how LASR, in combination with mutagenesis, can be used to estimate the yields of competing pathways, as well as to generate and characterize transient, unstable complexes.

Thermodynamic and Kinetic Analysis of an RNA Kissing Interaction and Its Resolution into an Extended Duplex

Kissing hairpin interactions form when the loop residues of two hairpins have Watson-Crick complementarity. In a unimolecular context, kissing interactions are important for tertiary folding and pseudoknot formation, whereas in a bimolecular context, they provide a basis for molecular recognition. In some cases, kissing complexes can be a prelude to strand displacement reactions where the two hairpins resolve to form a stable extended intermolecular duplex. The kinetics and thermodynamics of kissing-complex formation and their subsequent strand-displacement reactions are poorly understood. Here, biophysical techniques including isothermal titration calorimetry, surface plasmon resonance, and single-molecule fluorescence have been employed to probe the factors that govern the stability of kissing complexes and their subsequent structural rearrangements. We show that the general understanding of RNA duplex formation can be extended to kissing complexes but that kissing complexes display an unusual level of stability relative to simple duplexes of the same sequence. These interactions form and break many times at room temperature before becoming committed to a slow, irreversible forward transition to the strand-displaced form. Furthermore, using smFRET we show that the primary difference between stable and labile kissing complexes is based almost completely on their off rates. Both stable and labile complexes form at the same rate within error, but less stable species dissociate rapidly, allowing us to understand how these complexes can help generate specificity along a folding pathway or during a gene regulation event.

Protein–RNA Dynamics in the Central Junction Control 30S Ribosome Assembly

Interactions between ribosomal proteins (rproteins) and ribosomal RNA (rRNA) facilitate the formation of functional ribosomes. S15 is a central domain primary binding protein that has been shown to trigger a cascade of conformational changes in 16S rRNA, forming the functional structure of the central domain. Previous biochemical and structural studies in vitro have revealed that S15 binds a three-way junction of helices 20, 21, and 22, including nucleotides 652-654 and 752-754. All junction nucleotides except 653 are highly conserved among the Bacteria. To identify functionally important motifs within the junction, we subjected nucleotides 652-654 and 752-754 to saturation mutagenesis and selected and analyzed functional mutants. Only 64 mutants with greater than 10% ribosome function in vivo were isolated. S15 overexpression complemented mutations in the junction loop in each of the partially active mutants, although mutations that produced inactive ribosomes were not complemented by overexpression of S15. Single-molecule Förster or fluorescence resonance energy transfer (smFRET) was used to study the Mg(2+)- and S15-induced conformational dynamics of selected junction mutants. Comparison of the structural dynamics of these mutants with the wild type in the presence and absence of S15 revealed specific sequence and structural motifs in the central junction that are important in ribosome function.

Protein-RNA Dynamics in the Central Junction Control 30S Ribosome Assembly

Interactions between ribosomal proteins and ribosomal RNA (rRNA) facilitate the formation of functional ribosomes. S15 is a central domain primary binding protein that has been shown to trigger a cascade of conformational changes in 16S rRNA, which result in the functional structure of the central domain. Previous biochemical and structural studies in vitro have revealed that S15 binds a three-way junction of helices 20, 21 and 22, including nucleotides 652-654 and 752-754. All junction nucleotides except 653 are highly conserved in bacteria implying that these nucleotides are functionally important. To identify functionally important sequences and structural elements within the junction, nucleotides 652-654 and 752-754 were subjected to saturation mutagenesis and functional mutants were selected and analyzed. Only 74 mutants were isolated with greater than 4% ribosome function in vivo. To determine whether S15 binding was affected by mutations in the junction loop, S15 was cloned and over-expressed with the junction mutants. S15 complemented mutations in the junction loop in each of the partially functional mutants. Nonfunctional mutants were not complemented by over-expression of S15.We used single molecule Fluorescence Resonance Energy Transfer (smFRET) to study the Mg2+ and S15 induced conformational changes occurred for all types of mutants and WT loops. Comparison of the structural dynamics of these mutants and WT sequence in the presence and absence of S15 revealed specific sequence and structural motifs in the junction loop that are important in ribosome function.