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 (K ∼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.

A divalent cation-dependent variant of the ribozyme with stringent Ca 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 Ca in intracellular signaling, engineered ribozymes with stringent Ca selectivity would be important in biotechnology. The wild-type ribozyme () requires glucosamine-6-phosphate (GlcN6P) as a catalytic cofactor.

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).

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.

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.

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.

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.