RNA-catalysed nucleotide synthesis

Unrau, Peter J.; Bartel, David P.

doi:10.1038/26193

Cited by 273 publications

(182 citation statements)

References 27 publications

Supporting

Mentioning

177

Contrasting

Unclassified

Order By: Relevance

“…The double-stranded DNA pool was constructed from two 110-nt DNA fragments, each containing 76 completely randomized positions (14). Each fragment was amplified separately in 110 ml PCR reactions and ligated through BanI sites to generate a final pool with the sequence: TTCTAATACGACTCACTATAG-GACAGCTCCGAGCATTCTCGTGTAGCTCTGACCTTGG-N 76 -GGCACC-N 76 -ACGCACATCGCAGCAAC (italics, T7 promoter; underlined nucleotides, TAR site; -N 76 -, 76 nucleotide random-sequence segment).…”

Section: Methodsmentioning

confidence: 99%

A ribozyme that ligates RNA to protein

Baskerville

Bartel

2002

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

We have used a combination of in vitro selection and rational design to generate ribozymes that form a stable phosphoamide bond between the 5 terminus of an RNA and a specific polypeptide. This reaction differs from that of previously identified ribozymes, although the product is analogous to the enzymenucleotidyl intermediates isolated during the reactions of certain proteinaceous enzymes, such as guanyltransferase, DNA ligase, and RNA ligase. Comparative sequence analysis of the isolated ribozymes revealed that they share a compact secondary structure containing six stems arranged in a four-helix junction and branched pseudoknot. An optimized version of the ribozyme reacts with substrate-fusion proteins, allowing it to be used to attach RNA tags to proteins both in vitro and within bacterial cells, suggesting a simple way to tag a specific protein with amplifiable information.in vitro selection ͉ nucleic acid tag ͉ phosphoamide ͉ Tat ͉ TAR I n vitro selection has been used in combination with rational design to identify and optimize numerous ribozymes and binding motifs (1-3). The results of these experiments continue to give insight into the capacity of RNA and DNA to catalyze a variety of chemical reactions and the ability of nucleic acids to recognize an array of substrates. Some of these catalysts are being developed into effective tools for use in molecular biology. These include allosteric ribozymes, used as detectors of small molecules and proteins, and DNA enzymes that efficiently cleave target sequences (4-6).Here we use in vitro selection coupled with rational design to produce a new ribozyme that ligates RNA to protein through the formation of a phosphoamide bond. This is similar to a reaction found in naturally occurring proteins such as guanyltransferase, and DNA and RNA ligases that form phosphoamide bonds as enzyme-nucleotidyl intermediates during cap formation and ligation events (7-9). The selection of this new ribozyme expands the repertoire of RNA-catalyzed chemistry and further illustrates the ability of RNA to chemically modify a variety of substrates. Moreover, this ribozyme might be used to synthesize oligonucleotide-tagged proteins in vitro and in vivo. These attached oligonucleotides might be useful as affinity tags or could function as amplifiable identifying markers. For example, this ribozyme might be used to attach mRNAs to the proteins that they encode. A diverse library of mRNA-protein fusions could be used in conjunction with in vitro selection strategies to identify previously undescribed proteins with unique or enhanced properties, as has been shown previously by using phage display, ribosome display, and mRNA display (10-13). Materials and MethodsPool Construction. The double-stranded DNA pool was constructed from two 110-nt DNA fragments, each containing 76 completely randomized positions (14). Each fragment was amplified separately in 110 ml PCR reactions and ligated through BanI sites to generate a final pool with the sequence: TTCTAATACGACTCACTATAG-GACAGCTCCGAGCATTCTCGTGT...

show abstract

Section: Methodsmentioning

confidence: 99%

A ribozyme that ligates RNA to protein

Baskerville

Bartel

2002

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…On the one hand, an RNA that catalyzes the racemization of a sterically hindered biphenyl is a typical and interesting example of a ribozyme that brings about a reaction that has no obvious relation to biochemistry (Prudent et al, 1994). On the other, the RNA-catalyzed synthesis of 4-thiouridine-5 -phosphate from 4-thiouracil and 5-phosphoribosyl-1-pyrophosphate is typical of the type of riboenzymatic reaction that, according to the RNA-first scenario, might have evolved when the availability of prebiotic nucleotides declined (Unrau & Bartel, 1998). However, at the very beginning of the evolution of the RNA World only one function was essential, namely catalysis of RNA replication.…”

Section: Ribozymesmentioning

confidence: 99%

Prebiotic Chemistry and the Origin of the RNA World

Orgel

2004

Critical Reviews in Biochemistry and Molecular Biology

909

248

View full text Add to dashboard Cite

The demonstration that ribosomal peptide synthesis is a ribozyme-catalyzed reaction makes it almost certain that there was once an RNA World. The central problem for origin-of-life studies, therefore, is to understand how a protein-free RNA World became established on the primitive Earth. We first review the literature on the prebiotic synthesis of the nucleotides, the nonenzymatic synthesis and copying of polynucleotides, and the selection of ribozyme catalysts of a kind that might have facilitated polynucleotide replication. This leads to a brief outline of the Molecular Biologists' Dream, an optimistic scenario for the origin of the RNA World. In the second part of the review we point out the many unresolved problems presented by the Molecular Biologists' Dream. This in turn leads to a discussion of genetic systems simpler than RNA that might have "invented" RNA. Finally, we review studies of prebiotic membrane formation.

show abstract

“…RNA enzymes have been described that catalyze the reverse reaction, the synthesis of a nucleoside from an activated sugar and a base (21). In contemporary biochemistry, however, the reactions of nucleoside biosynthesis and degradation are catalyzed by protein enzymes.…”

Section: Discussionmentioning

confidence: 99%

“…In view of the mechanistic similarities of N-glycosylase proteins and the likely mechanism of the nucleoside synthase ribozyme (21) it is reasonable to propose that the 10-28 Nglycosylase DNA enzyme catalyzes depurination by specific recognition of the target deoxyguanosine, protonation of the N7 position, and delivery of a water molecule to the C1Ј position. The DNA enzyme is specific for deoxyguanosine, although the way in which it recognizes this residue is not known.…”

Section: Discussionmentioning

confidence: 99%

A DNA enzyme with N -glycosylase activity

Sheppard

Ordoukhanian

Joyce

2000

Proc. Natl. Acad. Sci. U.S.A.

125

View full text Add to dashboard Cite

In vitro evolution was used to develop a DNA enzyme that catalyzes the site-specific depurination of DNA with a catalytic rate enhancement of about 10 6 -fold. The reaction involves hydrolysis of the N-glycosidic bond of a particular deoxyguanosine residue, leading to DNA strand scission at the apurinic site. The DNA enzyme contains 93 nucleotides and is structurally complex. It has an absolute requirement for a divalent metal cation and exhibits optimal activity at about pH 5. The mechanism of the reaction was confirmed by analysis of the cleavage products by using HPLC and mass spectrometry. The isolation and characterization of an Nglycosylase DNA enzyme demonstrates that single-stranded DNA, like RNA and proteins, can form a complex tertiary structure and catalyze a difficult biochemical transformation. This DNA enzyme provides a new approach for the site-specific cleavage of DNA molecules.depurination ͉ DNA cleavage ͉ DNA repair ͉ in vitro evolution ͉ nucleic acid catalysis A lthough the sugar-phosphate backbone of DNA is highly resistant to hydrolysis, DNA is not impervious to degradation by other means. Damage can occur to either the sugar or nucleobase components of DNA, compromising its strand continuity and information content. Various environmental factors, such as high temperature, oxidative conditions, ionizing radiation, and reactive chemical agents, all can lead to alteration of DNA (1). One of the simplest and best studied forms of DNA damage is spontaneous depurination. This reaction involves hydrolytic cleavage of the N-glycosidic bond of a purine nucleoside ( Fig. 1), giving rise to an apurinic (AP) site (compound 1). The reaction is catalyzed by acid and facilitated by heating. It proceeds by a S N 1-like loss of the purine to give an oxocarbenium ion, which subsequently is trapped with water.Production of AP sites in DNA can result in the loss of genetic information, both because AP sites themselves are mutagenic (2, 3) and because these sites can lead to DNA strand scission (4). As illustrated in Fig. 1, hydrolysis of the N-glycosidic bond unmasks the latent aldehyde functionality at the C1Ј position, rendering the 3Ј-phosphate group susceptible to ␤-elimination (compound 2). It has been estimated that about 10,000 AP sites are produced in a typical mammalian cell each day (5, 6). Not surprisingly, organisms have evolved elaborate biochemical pathways to repair and͞or minimize the effects of these lesions (7-9).Although the primary role of DNA is information storage, it also has structural and functional properties. In recent years, the laboratory evolution of catalytic DNA molecules has demonstrated that single-stranded DNA is capable of forming tertiary structural motifs that catalyze a variety of chemical transformations with rate enhancements comparable to those of naturally occurring enzymes (10, 11). It is reasonable to suppose that more complex DNA enzymes could be developed, including ones that function analogously to DNA repair enzymes.Starting with a population of 10 14 random-seq...

show abstract

RNA-catalysed nucleotide synthesis

Cited by 273 publications

References 27 publications

A ribozyme that ligates RNA to protein

A ribozyme that ligates RNA to protein

Prebiotic Chemistry and the Origin of the RNA World

A DNA enzyme with N -glycosylase activity

Contact Info

Product

Resources

About