Genetically encoded fluorescent protein tags revolutionized proteome studies, while 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, is one of the most red-shifted 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 make 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.

Diverse small molecules interact with catalytic RNAs (ribozymes) as substrates and cofactors, and their intracellular concentrations are sensed by gene-regulatory mRNA domains (riboswitches) that modulate transcription, splicing, translation, or RNA stability. Although recognition mechanisms vary from RNA to RNA, structural analyses reveal recurring strategies that arise from the intrinsic properties of RNA such as base pairing and stacking with conjugated heterocycles, and cation-dependent recognition of anionic functional groups. These studies also suggest that, to a first approximation, the magnitude of ligand-induced reorganization of an RNA is inversely proportional to the complexity of the riboswitch or ribozyme. How these small molecule bindinginduced changes in RNA lead to alteration in gene expression is less well understood. While different riboswitches have been proposed to be under either kinetic or thermodynamic control, the biochemical and structural mechanisms that give rise to regulatory consequences downstream of small molecule recognition by RNAs mostly remain to be elucidated.Ribozymes and riboswitches starkly demonstrate the ability of RNA to fold into complex structures that position functional groups with exquisite precision. The former are catalytic RNAs; the latter, cis-acting regulatory mRNA domains that respond to the intracellular concentration of small molecule metabolites and second messengers [the first example of a trans-acting riboswitch RNA was recently described (1)]. In vitro, both ribozymes and riboswitches can function in the absence of protein cofactors, although some catalytic RNAs are known to require chaperones [reviewed in (2)] for in vivo activity, and riboswitches ultimately need to interface with the rest of the gene expression (transcription, splicing, translation, or RNA degradation) machinery for their small molecule-dependent regulatory activity to become manifest. Over the past decade, structural analyses have shed light on the mechanism of small molecule recognition by ribozymes (as substrates and coenzymes) and riboswitches (as regulatory signals). We review the state of knowledge of small molecule recognition by RNA, and how small molecule binding gives rise to genetic regulation. Ligand recognition by natural and artificial aptamers Structures of riboswitch aptamer domainsAt the time of writing, structures of the ligand-binding [or "aptamer" (3) reported: cyclic diguanylate (c-di-GMP) (4,5), flavin mononucleotide (FMN) (6), glmS (7,8), lysine (9,10), preQ 1 (11-13), magnesium ion (14), purine (guanine and adenine) (15,16), S-adenosylmethionine Class I (SAM-I) (17), SAM-II (18), , and thiamine pyrophosphate (TPP) (20)(21)(22). The aptamer domains of these riboswitches all adopt distinctly different structures. There are no discernible evolutionary relationships between different classes of riboswitches, making it uncertain whether each class descends from an independent evolutionary ancestor, or if sequence and structure divergence have proceeded too f...

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.

Uniquely among known ribozymes, the glmS ribozyme-riboswitch requires a small-molecule coenzyme, glucosamine-6-phosphate (GlcN6P). Although consistent with its gene-regulatory function, use of GlcN6P is unexpected because all other characterized self-cleaving ribozymes employ 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 wildtype fold but employs divalent cations for catalysis and is insensitive to GlcN6P. Biochemical and crystallographic comparisons of wildtype 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 novel biochemical activities on structured RNAs. Thus, families of structurally related ribozymes with divergent function may exist.

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.