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R R Breaker

Publications and source records attributed to R R Breaker.

7 recordsLinked to original sources
PubMed1995

Self-incorporation of coenzymes by ribozymes.

RNA molecules that are assembled from the four standard nucleotides contain a limited number of chemical functional groups, a characteristic that is generally thought to restrict the potential for catalysis by ribozymes. Although polypeptides carry a wider range of functional groups, many contemporary protein-based enzymes employ coenzymes to augment their capabilities. The coenzymes possess additional chemical moieties that can participate directly in catalysis and thereby enhance catalytic function. In this work, we demonstrate a mechanism by which ribozymes can supplement their limited repertoire of functional groups through RNA-catalyzed incorporation of various coenzymes and coenzyme analogues. The group I ribozyme of Tetrahymena thermophila normally mediates a phosphoester transfer reaction that results in the covalent attachment of guanosine to the ribozyme. Here, a shortened version of the ribozyme is shown to catalyze the self-incorporation of coenzymes and coenzyme analogues, such as NAD+ and dephosphorylated CoA-SH. Similar ribozyme activities may have played an important role in the "RNA world," when RNA enzymes are thought to have maintained a complex metabolism in the absence of proteins and would have benefited from the inclusion of additional functional groups.

Animals↗
PubMed1994

Continuous in vitro evolution of bacteriophage RNA polymerase promoters.

Rapid in vitro evolution of bacteriophage T7, T3, and SP6 RNA polymerase promoters was achieved by a method that allows continuous enrichment of DNAs that contain functional promoter elements. This method exploits the ability of a special class of nucleic acid molecules to replicate continuously in the presence of both a reverse transcriptase and a DNA-dependent RNA polymerase. Replication involves the synthesis of both RNA and cDNA intermediates. The cDNA strand contains an embedded promoter sequence, which becomes converted to a functional double-stranded promoter element, leading to the production of RNA transcripts. Synthetic cDNAs, including those that contain randomized promoter sequences, can be used to initiate the amplification cycle. However, only those cDNAs that contain functional promoter sequences are able to produce RNA transcripts. Furthermore, each RNA transcript encodes the RNA polymerase promoter sequence that was responsible for initiation of its own transcription. Thus, the population of amplifying molecules quickly becomes enriched for those templates that encode functional promoters. Optimal promoter sequences for phage T7, T3, and SP6 RNA polymerase were identified after a 2-h amplification reaction, initiated in each case with a pool of synthetic cDNAs encoding greater than 10(10) promoter sequence variants.

Bacteriophage T3↗
PubMed1994

Emergence of a replicating species from an in vitro RNA evolution reaction.

The technique of self-sustained sequence replication allows isothermal amplification of DNA and RNA molecules in vitro. This method relies on the activities of a reverse transcriptase and a DNA-dependent RNA polymerase to amplify specific nucleic acid sequences. We have modified this protocol to allow selective amplification of RNAs that catalyze a particular chemical reaction. During an in vitro RNA evolution experiment employing this modified system, a unique class of "selfish" RNAs emerged and replicated to the exclusion of the intended RNAs. Members of this class of selfish molecules, termed RNA Z, amplify efficiently despite their inability to catalyze the target chemical reaction. Their amplification requires the action of both reverse transcriptase and RNA polymerase and involves the synthesis of both DNA and RNA replication intermediates. The proposed amplification mechanism for RNA Z involves the formation of a DNA hairpin that functions as a template for transcription by RNA polymerase. This arrangement links the two strands of the DNA, resulting in the production of RNA transcripts that contain an embedded RNA polymerase promoter sequence.

Avian Myeloblastosis Virus↗
PubMed1994

Production of RNA by a polymerase protein encapsulated within phospholipid vesicles.

Catalyzed polymerization reactions represent a primary anabolic activity of all cells. It can be assumed that early cells carried out such reactions, in which macromolecular catalysts were encapsulated within some type of boundary membrane. In the experiments described here, we show that a template-independent RNA polymerase (polynucleotide phosphorylase) can be encapsulated in dimyristoyl phosphatidylcholine vesicles without substrate. When the substrate adenosine diphosphate (ADP) was provided externally, long-chain RNA polymers were synthesized within the vesicles. Substrate flux was maximized by maintaining the vesicles at the phase transition temperature of the component lipid. A protease was introduced externally as an additional control. Free enzyme was inactivated under identical conditions. RNA products were visualized in situ by ethidium bromide fluorescence. The products were harvested from the liposomes, radiolabeled, and analyzed by polyacrylamide gel electrophoresis. Encapsulated catalysts represent a model for primitive cellular systems in which an RNA polymerase was entrapped within a protected microenvironment.

Adenosine Diphosphate↗
PubMed1994

Inventing and improving ribozyme function: rational design versus iterative selection methods.

Two major strategies for generating novel biological catalysts exist. One relies on our knowledge of biopolymer structure and function to aid in the 'rational design' of new enzymes. The other, often called 'irrational design', aims to generate new catalysts, in the absence of detailed physicochemical knowledge, by using selection methods to search a library of molecules for functional variants. Both strategies have been applied, with considerable success, to the remodeling of existing ribozymes and the development of ribozymes with novel catalytic function. The two strategies are by no means mutually exclusive, and are best applied in a complementary fashion to obtain ribozymes with the desired catalytic properties.

Animals↗
PubMed1993

Synthesis and properties of adenosine oligonucleotide analogues containing methylene groups in place of phosphodiester 5'-oxygens.

The ADP analogue in which the 5'-oxygen has been replaced by a methylene group can be prepared by condensing 5'-deoxy-5'-phosphonomethyladenosine with inorganic phosphate. This analogue readily polymerizes onto the primer A-A in the presence of the enzyme polynucleotide phosphorylase and either Mg2+ or Mn2+. The initial products are of the form A-A(-cA)n-cA (where "-" and "-c" stand for the normal phosphodiester linkage and the linkage in which the 5'-oxygen is replaced with the methylene group, respectively). Treatment of these with alkali yields adenosine 2'(3')-phosphate and the series (A(-cA)n-cA containing only phosphonomethylene linkages. The decamer A(-cA)8-cA interacts with two molecules of U(-U)8-U to form a triple-standard structure that has a stability similar to that exhibited by the analogous complex formed from A(-A)8-A and U(-U)8-U. This property, along with the resistance of these oligomer analogues toward nucleases that cleave phosphodiester linkages between the phosphorus and the 5'-oxygen, should provide a strong rationale for application of phosphonomethylene linkages in schemes for therapeutic drug design that use the antisense strategy.

Adenosine Diphosphate↗