Architectures and complex functions of tandem riboswitches.

Riboswitch architectures that involve the binding of a single ligand to a single RNA aptamer domain result in ordinary dose-response curves that require approximately a 100-fold change in ligand concentration to cover nearly the full dynamic range for gene regulation. However, by using multiple riboswitches or aptamer domains in tandem, these ligand-sensing structures can produce additional, complex gene control outcomes. In the current study, we have computationally searched for tandem riboswitch architectures in bacteria to provide a more complete understanding of the diverse biological and biochemical functions of gene control elements that are made exclusively of RNA. Numerous different arrangements of tandem homologous riboswitch architectures are exploited by bacteria to create more 'digital' gene control devices, which operate over a narrower ligand concentration range. Also, two heterologous riboswitch aptamers are sometimes employed to create two-input Boolean logic gates with various types of genetic outputs. These findings illustrate the sophisticated genetic decisions that can be made by using molecular sensors and switches based only on RNA.

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. brucei PPR protein (KRIPP11) using electrophoretic mobility shift assays, fluorescence anisotropy, circular dichroism spectroscopy, and in vitro selection. We found KRIPP11 to be an RNA binding protein with specificity for sequences of four or more consecutive guanosine residues (G-tracts). Such G-tracts are dramatically enriched in T. brucei mitochondrial transcripts that are destined for extensive uridine insertion/deletion editing but are not present in mRNAs following editing. We further found that the quadruplex oligoguanosine RNA conformation is preferentially recognized by KRIPP11 over other conformational forms, and is bound without disruption of the quadruplex structure. In combination with prior data demonstrating association of KRIPP11 with the small ribosomal subunit, these results suggest possible roles for KRIPP11 in bridging mRNA maturation and translation or in facilitating translation of unusual dual-coded open reading frames.

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 North-methanocarbacytidine modification that prevents fluctuations to C2'-endo conformations while maintaining an intact 2'-hydroxyl nucleophile. Spin-relaxation data demonstrate that the active-site Cyt-6 undergoes conformational exchange attributed to sampling of a minor C2'-endo state with an exchange lifetime on the order of microseconds to tens of microseconds. A conformationally restricted species in which the fluctuations to the minor species are interrupted shows a drastic decrease in self-cleavage activity. Taken together, these data indicate that dynamic sampling of a minor species at the active site of this ribozyme, and likely of related naturally occurring motifs, is strongly coupled to catalytic function. The combination of NMR dynamics analysis with functional probing via conformational restriction is a general methodology for dissecting dynamics-function relationships in RNA.

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. We find docking to be strongly enthalpy-driven and to be accompanied by substantial activation barriers for association and dissociation, consistent with the structural reorganization of both internal loops upon complex formation. Comparisons with the parallel analysis of a ribozyme variant carrying a 2'-O-methyl modification at the self-cleavage site and with published data in other systems reveal a surprising diversity of thermodynamic signatures, emphasizing the delicate balance of contributions to the free energy of formation of RNA tertiary structure.

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. The very slow on-rate for this relatively tight interaction has led us to hypothesize a double conformational capture mechanism for RNA-RNA recognition. We used extensive molecular dynamics simulations to assess conformational sampling in the undocked form of the loop domain containing the scissile phosphate (loop A). We observed several major accessible conformations with distinctive patterns of hydrogen bonding and base stacking interactions in the active-site internal loop. Several important conformational features characteristic of the docked state were observed in well-populated substates, consistent with the kinetic sampling of docking-competent states by isolated loop A. Our observations suggest a hybrid or multistage binding mechanism, in which initial conformational selection of a docking-competent state is followed by induced-fit adjustment to an in-line, chemically reactive state only after formation of the initial complex with loop B.

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. We pay particular attention to practical aspects of these studies, such as careful preparation of samples with fixed free concentrations of cations in order to avoid errors due to ion depletion effects that are common in RNA systems. Examples of applications from our own work with the hairpin ribozyme are shown. Distinctions among the data handling procedures for the various techniques used and solution conditions encountered are also discussed.

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. We investigate the thermodynamics and kinetics of the docking process using constructs in which loop A and loop B reside on separate molecules. Using a novel CD difference assay to isolate the effects of metal ions linked to domain docking, we find the intermolecular docking process to be driven by sub-millimolar concentrations of the exchange-inert Co(NH 3) 6 (3+). RNA self-cleavage requires binding of lower-affinity ions with greater apparent cooperativity than the docking process itself, implying that, even in the absence of direct coordination to RNA, metal ions play a catalytic role in hairpin ribozyme function beyond simply driving loop-loop docking. Surface plasmon resonance assays reveal remarkably slow molecular association, given the relatively tight loop-loop interaction. This observation is consistent with a "double conformational capture" model in which only collisions between loop A and loop B molecules that are simultaneously in minor, docking-competent conformations are productive for binding.

A comparison of plotless density estimators using Monte Carlo simulation on totally enumerated field data sets.

BACKGROUND: Plotless density estimators are those that are based on distance measures rather than counts per unit area (quadrats or plots) to estimate the density of some usually stationary event, e.g. burrow openings, damage to plant stems, etc. These estimators typically use distance measures between events and from random points to events to derive an estimate of density. The error and bias of these estimators for the various spatial patterns found in nature have been examined using simulated populations only. In this study we investigated eight plotless density estimators to determine which were robust across a wide range of data sets from fully mapped field sites. They covered a wide range of situations including animal damage to rice and corn, nest locations, active rodent burrows and distribution of plants. Monte Carlo simulations were applied to sample the data sets, and in all cases the error of the estimate (measured as relative root mean square error) was reduced with increasing sample size. The method of calculation and ease of use in the field were also used to judge the usefulness of the estimator. Estimators were evaluated in their original published forms, although the variable area transect (VAT) and ordered distance methods have been the subjects of optimization studies. RESULTS: An estimator that was a compound of three basic distance estimators was found to be robust across all spatial patterns for sample sizes of 25 or greater. The same field methodology can be used either with the basic distance formula or the formula used with the Kendall-Moran estimator in which case a reduction in error may be gained for sample sizes less than 25, however, there is no improvement for larger sample sizes. The variable area transect (VAT) method performed moderately well, is easy to use in the field, and its calculations easy to undertake. CONCLUSION: Plotless density estimators can provide an estimate of density in situations where it would not be practical to layout a plot or quadrat and can in many cases reduce the workload in the field.