A Promiscuous Ribozyme Promotes Nucleotide Synthesis in Addition to Ribose Chemistry (original) (raw)
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Current Opinion in Structural Biology, 1997
The number of RNA molecules that have novel catalytic activities has dramatically increased during the past two years. This ribozymic boom is not due to the discovery of additional examples of natural ribozymes but rather to the development of artificial ribozymes isolated by in vitro selection and evolution techniques. The structural and functional complexities of these artificial ribozymes, however, do not match those of the larger natural ribozymes. The understanding of both RNA structure and catalysis performed by natural and artificial ribozymes paves the way for the creation of RNA molecules that are able to efficiently catalyze more complex reactions.
RNA-catalysed nucleotide synthesis
Nature, 1998
The 'RNA world' hypothesis proposes that early life developed by making use of RNA molecules, rather than proteins, to catalyse the synthesis of important biological molecules. It is thought, however, that the nucleotides constituting RNA were scarce on early Earth. RNA-based life must therefore have acquired the ability to synthesize RNA nucleotides from simpler and more readily available precursors, such as sugars and bases. Plausible prebiotic synthesis routes have been proposed for sugars, sugar phosphates and the four RNA bases, but the coupling of these molecules into nucleotides, specifically pyrimidine nucleotides, poses a challenge to the RNA world hypothesis. Here we report the application of in vitro selection to isolate RNA molecules that catalyse the synthesis of a pyrimidine nucleotide at their 3' terminus. The finding that RNA can catalyse this type of reaction, which is modelled after pyrimidine synthesis in contemporary metabolism, supports the idea of a...
RNA synthesis by in vitro selected ribozymes for recreating an RNA world
Life (Basel, Switzerland), 2015
The RNA world hypothesis states that during an early stage of life, RNA molecules functioned as genome and as the only genome-encoded catalyst. This hypothesis is supported by several lines of evidence, one of which is the in vitro selection of catalytic RNAs (ribozymes) in the laboratory for a wide range of reactions that might have been used by RNA world organisms. This review focuses on three types of ribozymes that could have been involved in the synthesis of RNA, the core activity in the self-replication of RNA world organisms. These ribozyme classes catalyze nucleoside synthesis, triphosphorylation, and the polymerization of nucleoside triphosphates. The strengths and weaknesses regarding each ribozyme's possible function in a self-replicating RNA network are described, together with the obstacles that need to be overcome before an RNA world organism can be generated in the laboratory.
Natural and unnatural ribozymes: back to the primordial RNA world
Research in microbiology, 2009
We review natural and in vitro selected ribozymes, for which combined studies could provide us with both insight into the functions performed by ancient RNA molecules in a primitive RNA world and a hypothesis about evolutionary steps that led to the contemporary world.
Thirty-five years of research into ribozymes and nucleic acid catalysis: where do we stand today?
F1000Research, 2016
Since the discovery of the first catalytic RNA in 1981, the field of ribozyme research has developed from the discovery of catalytic RNA motifs in nature and the elucidation of their structures and catalytic mechanisms, into a field of engineering and design towards application in diagnostics, molecular biology and medicine. Owing to the development of powerful protocols for selection of nucleic acid catalysts with a desired functionality from random libraries, the spectrum of nucleic acid supported reactions has greatly enlarged, and importantly, ribozymes have been accompanied by DNAzymes. Current areas of research are the engineering of allosteric ribozymes for artificial regulation of gene expression, the design of ribozymes and DNAzymes for medicinal and environmental diagnostics, and the demonstration of RNA world relevant ribozyme activities. In addition, new catalytic motifs or novel genomic locations of known motifs continue to be discovered in all branches of life by the h...
Multiple translational products from a five-nucleotide ribozyme
Proceedings of the National Academy of Sciences, 2010
An indispensable step in protein biosynthesis is the 2 0 ð3 0 Þ aminoacylation of tRNA by aminoacyl-tRNA synthetases. Here we show that a similar activity exists in a tiny, 5-nt-long RNA enzyme with a 3-nt active center. The small ribozyme initially trans-phenylalanylates a partially complementary 4-nt RNA selectively at its terminal 2 0 -ribose hydroxyl using PheAMP, the natural form for activated amino acid. The initial 2 0 Phe-RNA product can be elaborated into multiple peptidyl-RNAs. Reactions do not require divalent cations, and have limited dependence on monovalent cations. Small size and minimal requirements for regiospecific translational activity strongly support the hypothesis that minuscule RNA enzymes participated in early forms of translation.
Combinatorial minimization and secondary structure determination of a nucleotide synthase ribozyme
RNA, 2003
We previously isolated from random sequences ribozymes able to form a glycosidic linkage between a ribose sugar and 4-thiouracil in a reaction that mimics protein-catalyzed nucleotide synthesis. Here we report on two serial in vitro selection experiments that defined the core motif of one of the nucleotide synthase ribozymes and provided improved versions of this ribozyme. The first selection experiment started from a degenerate sequence pool based on the previously isolated sequence and used a selection-amplification protocol that allowed the sequence requirements at the 3 terminus of the ribozyme to be interrogated. Comparing the active sequences identified in this experiment revealed the complicated secondary structure of the nucleotide synthase ribozyme. A second selection was then performed to remove nonessential sequence from the ribozyme. This selection started with a pool with variation introduced in both the sequence and the length of the nonconserved loops and joining regions. This pool was generated using a partial reblocking/deblocking strategy on a DNA synthesizer, allowing the combinatorial synthesis of both point deletions and point substitutions. The consensus ribozyme motif that emerged was an ∼71 nt pseudoknot structure with five stems and two important joining segments. Comparative sequence analysis and a cross-linking experiment point to the probable location of nucleotide synthesis. The prototype isolate from the second selection was nearly 35 times more efficient than the initial isolate and at least 10 8 times more efficient than an upper limit of an as-yet undetectable uncatalyzed reaction, supporting the idea that RNA-catalyzed nucleotide synthesis might have been important in an RNA world.
Nucleobase-mediated general acid-base catalysis in the Varkud satellite ribozyme
Proceedings of the National Academy of Sciences, 2010
Existing evidence suggests that the Varkud satellite (VS) ribozyme accelerates the cleavage of a specific phosphodiester bond using general acid-base catalysis. The key functionalities are the nucleobases of adenine 756 in helix VI of the ribozyme, and guanine 638 in the substrate stem loop. This results in a bell-shaped dependence of reaction rate on pH, corresponding to groups with pK a ¼ 5.2 and 8.4. However, it is not possible from those data to determine which nucleobase is the acid, and which the base. We have therefore made substrates in which the 5′ oxygen of the scissile phosphate is replaced by sulfur. This labilizes the leaving group, removing the requirement for general acid catalysis. This substitution restores full activity to the highly impaired A756G ribozyme, consistent with general acid catalysis by A756 in the unmodified ribozyme. The pH dependence of the cleavage of the phosphorothiolatemodified substrates is consistent with general base catalysis by nucleobase at position 638. We conclude that cleavage of the substrate by the VS ribozyme is catalyzed by deprotonation of the 2′-O nucleophile by G638 and protonation of the 5′-O leaving group by A756.
Ribozyme Catalysis Revisited: Is Water Involved?
Molecular Cell, 2007
Enzymatic catalysis by RNA was discovered 25 years ago, yet mechanistic insights are emerging only slowly. Thought to be metalloenzymes at first, some ribozymes proved more versatile than anticipated when shown to utilize their own functional groups for catalysis. Recent evidence suggests that some may also judiciously place structural water molecules to shuttle protons in acid-base catalyzed reactions.