Structural basis for high-affinity fluorophore binding and activation by RNA Mango.

Genetically encoded fluorescent protein tags have revolutionized proteome studies, whereas the lack of intrinsically fluorescent RNAs has hindered transcriptome exploration. Among several RNA-fluorophore complexes that potentially address this problem, RNA Mango has an exceptionally high affinity for its thiazole orange (TO)-derived fluorophore, TO1-Biotin (Kd ∼3 nM), and, in complex with related ligands, it is one of the most redshifted fluorescent macromolecular tags known. To elucidate how this small aptamer exhibits such properties, which make it well suited for studying low-copy cellular RNAs, we determined its 1.7-Å-resolution co-crystal structure. Unexpectedly, the entire ligand, including TO, biotin and the linker connecting them, abuts one of the near-planar faces of the three-tiered G-quadruplex. The two heterocycles of TO are held in place by two loop adenines and form a 45° angle with respect to each other. Minimizing this angle would increase quantum yield and further improve this tool for in vivo RNA visualization.

A divalent cation-dependent variant of the glmS ribozyme with stringent Ca2+ selectivity co-opts a preexisting nonspecific metal ion-binding site.

Ribozymes use divalent cations for structural stabilization, as catalytic cofactors, or both. Because of the prominent role of Ca2+ in intracellular signaling, engineered ribozymes with stringent Ca2+ selectivity would be important in biotechnology. The wild-type glmS ribozyme (glmSWT) requires glucosamine-6-phosphate (GlcN6P) as a catalytic cofactor. Previously, a glmS ribozyme variant with three adenosine mutations (glmSAAA) was identified, which dispenses with GlcN6P and instead uses, with little selectivity, divalent cations as cofactors for site-specific RNA cleavage. We now report a Ca2+-specific ribozyme (glmSCa) evolved from glmSAAA that is >10,000 times more active in Ca2+ than Mg2+, is inactive in even 100 mM Mg2+, and is not responsive to GlcN6P. This stringent selectivity, reminiscent of the protein nuclease from Staphylococcus, allows rapid and selective ribozyme inactivation using a Ca2+ chelator such as EGTA. Because glmSCa functions in physiologically relevant Ca2+ concentrations, it can form the basis for intracellular sensors that couple Ca2+ levels to RNA cleavage. Biochemical analysis of glmSCa reveals that it has co-opted for selective Ca2+ binding a nonspecific cation-binding site responsible for structural stabilization in glmSWT and glmSAAA Fine-tuning of the selectivity of the cation site allows repurposing of this preexisting molecular feature.

Many Activities, One Structure: Functional Plasticity of Ribozyme Folds.

Catalytic RNAs, or ribozymes, are involved in a number of essential biological processes, such as replication of RNA genomes and mobile genetic elements, RNA splicing, translation, and RNA degradation. The function of ribozymes requires the formation of active sites decorated with RNA functional groups within defined three-dimensional (3D) structures. The genotype (sequence) of RNAs ultimately determines what 3D structures they adopt (as a function of their environmental conditions). These 3D structures, in turn, give rise to biochemical activity, which can further elaborate them by catalytic rearrangements or association with other molecules. The fitness landscape of a non-periodic linear polymer, such as RNA, relates its primary structure to a phenotype. Two major challenges in the analysis of ribozymes is to map all possible genotypes to their corresponding catalytic activity (that is, to determine their fitness landscape experimentally), and to understand whether their genotypes and three-dimensional structures can support multiple different catalytic functions. Recently, the combined results of experiments that employ in vitro evolution methods, high-throughput sequencing and crystallographic structure determination have hinted at answers to these two questions: while the fitness landscape of ribozymes is rugged, meaning that their catalytic activity cannot be optimized by a smooth trajectory in sequence space, once an RNA achieves a stable three-dimensional fold, it can be endowed with distinctly different biochemical activities through small changes in genotype. This functional plasticity of highly structured RNAs may be particularly advantageous for the adaptation of organisms to drastic changes in selective pressure, or for the development of new biotechnological tools.

In vitro evolution of coenzyme-independent variants from the glmS ribozyme structural scaffold.

Uniquely among known natural ribozymes that cleave RNA sequence-specifically, the glmS ribozyme-riboswitch employs a small molecule, glucosamine-6-phosphate (GlcN6P) as a catalytic cofactor. In vitro selection was employed to search for coenzyme-independent variants of this ribozyme. In addition to shedding light on the catalytic mechanism of the ribozyme, such variants could resemble the evolutionary ancestors of the modern, GlcN6P-regulated ribozyme-riboswitch. A mutant pool was constructed such that the secondary structure elements, which define the triply-pseudoknotted global fold of the ribozyme, was preserved. A stringent selection scheme that relies on thiol-mercury affinity chromatography for separating active and inactive sequences ultimately yielded a triple mutant with a cleavage rate exceeding 3min(-1) that only requires divalent cations for activity. Mutational analysis demonstrated that a point reversion of the variant toward the wild-type sequence was sufficient to partially restore GlcN6P-dependence, suggesting that coenzyme dependence can be readily be acquired by RNAs that adopt the glmS ribozyme fold. The methods employed to perform this selection experiment are described in detail in this review.

An in vitro evolved glmS ribozyme has the wild-type fold but loses coenzyme dependence.

Uniquely among known ribozymes, the glmS ribozyme-riboswitch requires a small-molecule coenzyme, glucosamine-6-phosphate (GlcN6P). Although consistent with its gene-regulatory function, the use of GlcN6P is unexpected because all of the other characterized self-cleaving ribozymes use RNA functional groups or divalent cations for catalysis. To determine what active site features make this ribozyme reliant on GlcN6P and to evaluate whether it might have evolved from a coenzyme-independent ancestor, we isolated a GlcN6P-independent variant through in vitro selection. Three active site mutations suffice to generate a highly reactive RNA that adopts the wild-type fold but uses divalent cations for catalysis and is insensitive to GlcN6P. Biochemical and crystallographic comparisons of wild-type and mutant ribozymes show that a handful of functional groups fine-tune the RNA to be either coenzyme or cation dependent. These results indicate that a few mutations can confer new biochemical activities on structured RNAs. Thus, families of structurally related ribozymes with divergent function may exist.

A promiscuous ribozyme promotes nucleotide synthesis in addition to ribose chemistry.

Here we report the in vitro selection of an unusual ribozyme that efficiently performs nucleotide synthesis even though it was selected to perform a distinctly different sugar chemistry. This ribozyme, called pR1, when derivatized with ribose 5-phosphate (PR) at its 3' terminus and incubated with 6-thioguanine, produces two interconverting thiol-containing products corresponding to a Schiff base and its Amadori rearranged product. Consistent with this hypothesis, removing the 2-hydroxyl from the PR substrate results in only a single product. Surprisingly, as this was not selected for, switching the tethered PR substrate to 5-phosphoribosyl 1-pyrophosphate results in the synthesis of 6-thioguanosine 5'-monophosphate. The discovery that a ribozyme can promote such distinct reactions spontaneously demonstrates that an RNA-mediated metabolism early in evolution could have evolved important new functionalities via ribozyme promiscuity.

Isolation of fast purine nucleotide synthase ribozymes.

Here we report the in vitro selection of fast ribozymes capable of promoting the synthesis of a purine nucleotide (6-thioguanosine monophosphate) from tethered 5-phosphoribosyl 1-pyrophosphate (PRPP) and 6-thioguanine ((6S)Gua). The two most proficient purine synthases have apparent efficiencies of 284 and 230 M(-1) min(-1) and are both significantly more efficient than pyrimidine nucleotide synthase ribozymes selected previously by a similar approach. Interestingly, while both ribozymes showed good substrate discrimination, one ribozyme had no detectable affinity for 6-thioguanine while the second had a K(m) of approximately 80 muM, indicating that these ribozymes use considerably different modes of substrate recognition. The purine synthases were isolated after 10 rounds of selection from two high-diversity RNA pools. The first pool contained a long random sequence region. The second pool contained random sequence elements interspersed with the mutagenized helical elements of a previously characterized 4-thiouridine synthase ribozyme. While nearly all of the ribozymes isolated from this biased pool population appeared to have benefited from utilizing one of the progenitor's helical elements, little evidence for more complicated secondary structure preservation was evident. The discovery of purine synthases, in addition to pyrimidine synthases, demonstrates the potential for nucleotide synthesis in an 'RNA World' and provides a context from which to study small molecule RNA catalysis.