Charge-driven condensation of RNA and proteins suggests broad role of phase separation in cytoplasmic environments.

Phase separation processes are increasingly being recognized as important organizing mechanisms of biological macromolecules in cellular environments. Well-established drivers of phase separation are multi-valency and intrinsic disorder. Here, we show that globular macromolecules may condense simply based on electrostatic complementarity.

Dynamics-Function Analysis in Catalytic RNA Using NMR Spin Relaxation and Conformationally Restricted Nucleotides.

A full understanding of biomolecular function requires an analysis of both the dynamic properties of the system of interest and the identification of those dynamics that are required for function. We describe NMR methods based on metabolically directed specific isotope labeling for the identification of molecular disorder and/or conformational transitions on the RNA backbone ribose groups. These analyses are complemented by the use of synthetic covalently modified nucleotides constrained to a single sugar pucker, which allow functional assessment of dynamics by selectively removing a minor conformer identified by NMR from the structural ensemble.

The 27 kDa Trypanosoma brucei Pentatricopeptide Repeat Protein is a G-tract Specific RNA Binding Protein.

Pentatricopeptide repeat (PPR) proteins, a helical repeat family of organellar RNA binding proteins, play essential roles in post-transcriptional RNA processing. In Trypanosoma brucei, an expanded family of PPR proteins localize to the parasite's single mitochondrion, where they are believed to perform important roles in both RNA processing and translation. We studied the RNA binding specificity of the simplest T.

Coupling between conformational dynamics and catalytic function at the active site of the lead-dependent ribozyme.

In common with other self-cleaving RNAs, the lead-dependent ribozyme (leadzyme) undergoes dynamic fluctuations to a chemically activated conformation. We explored the connection between conformational dynamics and self-cleavage function in the leadzyme using a combination of NMR spin-relaxation analysis of ribose groups and conformational restriction via chemical modification. The functional studies were performed with a -methanocarbacytidine modification that prevents fluctuations to C2'-endo conformations while maintaining an intact 2'-hydroxyl nucleophile.

Thermodynamics and kinetics of RNA tertiary structure formation in the junctionless hairpin ribozyme.

The hairpin ribozyme consists of two RNA internal loops that interact to form the catalytically active structure. This docking transition is a rare example of intermolecular formation of RNA tertiary structure without coupling to helix annealing. We have used temperature-dependent surface plasmon resonance (SPR) to characterize the thermodynamics and kinetics of RNA tertiary structure formation for the junctionless form of the ribozyme, in which loops A and B reside on separate molecules.

Intrinsic Base-Pair Rearrangement in the Hairpin Ribozyme Directs RNA Conformational Sampling and Tertiary Interface Formation.

Dynamic fluctuations in RNA structure enable conformational changes that are required for catalysis and recognition. In the hairpin ribozyme, the catalytically active structure is formed as an intricate tertiary interface between two RNA internal loops. Substantial alterations in the structure of each loop are observed upon interface formation, or docking.

Unraveling the thermodynamics and kinetics of RNA assembly: surface plasmon resonance, isothermal titration calorimetry, and circular dichroism.

The mechanisms and driving forces of the assembly of RNA tertiary structure are a topic of much current interest. In several systems, including our own work in the docking transition of the hairpin ribozyme, intramolecular RNA tertiary folding has been converted into an intermolecular binding event, allowing the full power of contemporary biophysical techniques to be brought to bear on the analysis. We review the use of three such methods: circular dichroism to isolate the binding of multivalent cations coupled to tertiary assembly, surface plasmon resonance to determine the rates of association and dissociation, and isothermal titration calorimetry to dissect the thermodynamic contributions to RNA assembly events.

Intermolecular domain docking in the hairpin ribozyme: metal dependence, binding kinetics and catalysis.

The hairpin ribozyme is a prototype small, self-cleaving RNA motif. It exists naturally as a four-way RNA junction containing two internal loops on adjoining arms. These two loops interact in a cation-driven docking step prior to chemical catalysis to form a tightly integrated structure, with dramatic changes occurring in the conformation of each loop upon docking.

Extensive backbone dynamics in the GCAA RNA tetraloop analyzed using 13C NMR spin relaxation and specific isotope labeling.

Conformational dynamics play a key role in the properties and functions of proteins and nucleic acids. Heteronuclear NMR spin relaxation is a uniquely powerful site-specific probe of dynamics in proteins and has found increasing applications to nucleotide base side chains and anomeric sites in RNA. Applications to the nucleic acid ribose backbone, however, have been hampered by strong magnetic coupling among ring carbons in uniformly 13C-labeled samples.

Conformationally restricted nucleotides as a probe of structure-function relationships in RNA.

We introduce the use of commercially available locked nucleic acids (LNAs) as a functional probe in RNA. LNA nucleotides contain a covalent linkage that restricts the pseudorotation phase of the ribose to C3'-endo (A-form). Introduction of an LNA at a single site thus allows the role of ribose structure and dynamics in RNA function to be assessed.

Structure-function relationships in RNA and RNP enzymes: recent advances.

The structural biology of ribozymes and ribonucleoprotein (RNP) enzymes is now sufficiently advanced that a true dialogue between structural and functional studies is possible. In this review, we consider three important systems in which an integration of structural and biochemical data has recently led to major advances in mechanistic understanding. In the hammerhead ribozyme, application-driven biochemical studies led to the discovery of a key structural interaction that had been omitted from previously-studied constructs.

Coordination environment of a site-bound metal ion in the hammerhead ribozyme determined by 15N and 2H ESEEM spectroscopy.

Although site-bound Mg2+ ions have been proposed to influence RNA structure and function, establishing the molecular properties of such sites has been challenging due largely to the unique electrostatic properties of the RNA biopolymer. We have previously determined that, in solution, the hammerhead ribozyme (a self-cleaving RNA) has a high-affinity metal ion binding site characterized by a K(d,app) < 10 microM for Mn2+ in 1 M NaCl and speculated that this site has functional importance in the ribozyme cleavage reaction. Here we determine both the precise location and the hydration level of Mn2+ in this site using ESEEM (electron spin-echo envelope modulation) spectroscopy.

Alternate-site isotopic labeling of ribonucleotides for NMR studies of ribose conformational dynamics in RNA.

Heteronuclear NMR spin relaxation studies of conformational dynamics are coming into increasing use to help understand the functions of ribozymes and other RNAs. Due to strong 13C-13C magnetic interactions within the ribose ring, however, these studies have thus far largely been limited to (13)C and (15)N resonances on the nucleotide base side chains. We report here the application of the alternate-site (13)C isotopic labeling scheme, pioneered by LeMaster for relaxation studies of amino acid side chains, to nucleic acid systems.

Water counting: quantitating the hydration level of paramagnetic metal ions bound to nucleotides and nucleic acids.

Binding of divalent metal ions plays a key role in the structure and function of ribozymes and other RNAs. In turn, the energetics and kinetics of the specific binding process are dominated by the balance between the cost of dehydrating the aqueous ion and the energy gained from inner-sphere interactions with the macromolecule. In this work, we introduce the use of the pulsed EPR technique of 2H Electron Spin-Echo Envelope Modulation (ESEEM) to determine the hydration level of Mn2+ ions bound to nucleotides and nucleic acids.

Structural analysis of metal ion ligation to nucleotides and nucleic acids using pulsed EPR spectroscopy.

Metal ions play key structural and functional roles in many nucleic acid systems, particularly as required cofactors for many catalytic RNA molecules (ribozymes). We apply the pulsed EPR technologies of electron spin-echo envelope modulation and electron spin-echo-electron nuclear double resonance to the structural analysis of the paramagnetic metal ion Mn(II) bound to nucleotides and nucleic acids. We demonstrate that pulsed EPR, supplemented with specific isotope labeling, can characterize ligation to nucleotide base nitrogens, outer-sphere interactions with phosphate groups, distances to sites of specific (2)H atom labels, and the hydration level of the metal ion.