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The noncoding RNA B2 and the RNA aptamer FC bind RNA polymerase (Pol) II and inhibit messenger RNA transcription initiation, but not elongation. We report the crystal structure of FC(*), the central part of FC RNA, bound to Pol II. FC(*) RNA forms a double stem-loop structure in the Pol II active center cleft. B2 RNA may bind similarly, as it competes with FC(*) RNA for Pol II interaction. Both RNA inhibitors apparently prevent the downstream DNA duplex and the template single strand from entering the cleft after DNA melting and thus interfere with open-complex formation. Elongation is not inhibited, as nucleic acids prebound in the cleft would exclude the RNA inhibitors. The structure also indicates that A-form RNA could interact with Pol II similarly to a B-form DNA promoter, as suggested for the bacterial transcription inhibitor 6S RNA.

Directed in vitro evolution can create RNA catalysts for a variety of organic reactions, supporting the "RNA world" hypothesis, which proposes that metabolic transformations in early life were catalyzed by RNA molecules rather than proteins. Among the most fundamental carbon-carbon bond-forming reactions in nature is the aldol reaction, mainly catalyzed by aldolases that utilize either an enamine mechanism (class I) or a Zn(2+) cofactor (class II). We report on isolation of a Zn(2+)-dependent ribozyme that catalyzes an aldol reaction at its own modified 5' end with a 4300-fold rate enhancement over the uncatalyzed background reaction. The ribozyme can also act as an intermolecular catalyst that transfers a biotinylated benzaldehyde derivative to the aldol donor substrate, coupled to an external hexameric RNA oligonucleotide, supporting the existence of RNA-originated biosynthetic pathways for metabolic sugar precursors and other biomolecules.

The ability to generate RNA molecules that can catalyze complex organic transformations not only facilitates the reconstruction and plausibility of possible prebiotic reaction pathways but is also crucial for elucidating the potential of the application of RNA catalysts in organic syntheses. Iterative RNA selection previously identified a ribozyme that catalyzes the Michael addition of a cysteine thiol to an alpha,beta-unsaturated amide. This reaction is chemically similar to the rate limiting step of the thymidylate synthase reaction, which is the corresponding reaction of a cysteine thiol to the double-bond of the uracil nucleobase. Here we provide a detailed description of the synthesis of the ribozyme substrates and the substrate oligonucleotides used for its characterization and the investigation of the background reaction. We also describe the further characterization of the ribozyme with respect to substrate specificity. We show that the thiol group of the cysteine nucleophile is essential for the reaction to proceed. When substituted for a thiomethyl group, no reaction takes place.

Background: In vitro selected ribozymes with nucleotide synthase, peptide and carbon-carbon bond forming activity provide insight into possible scenarios on how chemical transformations may have been catalyzed before protein enzymes had evolved. Metabolic pathways based on ribozymes may have existed at an early stage of evolution., Results: We have isolated a novel ribozyme that mediates Michael-adduct formation at a Michael-acceptor substrate, similar to the rate-limiting step of the mechanistic sequence of thymidylate synthase. The kinetic characterization of this catalyst revealed a rate enhancement by a factor of approximately 10(5). The ribozyme shows substrate specificity and can act as an intermolecular catalyst which transfers the Michael-donor substrate onto an external 20-mer RNA oligonucleotide containing the Michael-acceptor system., Conclusion: The ribozyme described here is the first example of a catalytic RNA with Michael-adduct forming activity which represents a key mechanistic step in metabolic pathways and other biochemical reactions. Therefore, previously unforeseen RNA-evolution pathways can be considered, for example the formation of dTMP from dUMP. The substrate specificity of this ribozyme may also render it useful in organic syntheses.