Nucleoside Phosphorothioates

Eckstein, Fritz

doi:10.1146/annurev.bi.54.070185.002055

Cited by 905 publications

(521 citation statements)

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“…Intriguingly, the steriospecificity of the thio effects matches that of protein-enzyme polymerases (20). Both the ribozyme and protein enzymes suffer mere elemental effects upon substitution of the pro-S P oxygen and much larger effects with the pro-R P substitution.…”

Section: Resultsmentioning

confidence: 89%

Recognition of Nucleoside Triphosphates during RNA-Catalyzed Primer Extension

et al. 2000

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In support of the idea that certain RNA molecules might be able to catalyze RNA replication, a ribozyme was previously generated that synthesizes short segments of RNA in a reaction modeled after that of proteinaceous RNA polymerases. Here, we describe substrate recognition by this polymerase ribozyme. Altering base or sugar moieties of the nucleoside triphosphate only moderately affects its utilization, provided that the alterations do not disrupt Watson-Crick pairing to the template. Correctly paired nucleotides have both a lower K m and a higher k cat , suggesting that differential binding and orientation each play roles in discriminating matched from mismatched nucleotides. Binding of the pyrophosphate leaving group appears weak, as evidenced by a very inefficient pyrophosphate-exchange reaction, the reverse of the primer-extension reaction. Indeed, substitutions at the γ-phosphate can be tolerated, although poorly. Thio substitutions of oxygen atoms at the reactive phosphate exert effects similar to those seen with cellular polymerases, leaving open the possibility of an active site analogous to those of protein enzymes. The polymerase ribozyme, derived from an efficient RNA ligase ribozyme, can achieve the very fast k cat of the parent ribozyme when the substrate of the polymerase (GTP) is replaced by an extended substrate (pppGGA), in which the GA dinucleotide extension corresponds to the second and third nucleotides of the ligase. This suggests that the GA dinucleotide, which had been deleted when converting the ligase into a polymerase, plays an important role in orienting the 5′-terminal nucleoside. Polymerase constructs that restore this missing orientation function should achieve much more efficient and perhaps more accurate RNA polymerization.The RNA world hypothesis proposes that early life relied on catalytic RNAs rather than protein enzymes for catalysis (1). Much of the appeal of this theory comes from the notion that ribozymes, which could have served as their own genes, would have been much simpler to duplicate than proteins (2-5). Hence, a vital component of the RNA world scenario is an RNA polymerase ribozyme responsible for replicating the essential ribozymes of early life (including itself). Whether there might be RNA sequences that can promote RNA polymerization with sufficient accuracy and efficiency for RNA self-replication is one of the fundamental unanswered questions related to the RNA world hypothesis (1, 6). Finding such a sequence would support the idea of the RNA world and provide a key component for the synthesis of simple living cells (7).The class I ligase ( Figure 1A) is a promising starting point for efforts to develop a self-replicating ribozyme. This ribozyme ligates two RNAs, generating a 3′,5′-phosphodiester linkage and releasing pyrophosphate, a reaction similar to that of RNA and DNA polymerases (8). Engineered forms of the ligase can polymerize short segments of RNA ( Figure 1C). In the presence of appropriate RNA templates and nucleoside triphosphates (NTP...

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Section: Resultsmentioning

confidence: 89%

Recognition of Nucleoside Triphosphates during RNA-Catalyzed Primer Extension

et al. 2000

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“…The Sp diastereomers of the NTPaS are incorporated into transcripts by T7 RNA polymerase with inversion of configuration at phosphorus (Eckstein, 1985;Griffith et al+, 1987)+ However, dNTPs and dNTPaS are not accepted as substrates+ Thus, for the incorporation of the dNTPaS the Y639F mutant had to be used, because it is known to accept dNTPs as well as certain 29-modified nucleotides much better as substrates than the wild type enzyme (Sousa & Padilla, 1995;Huang et al+, 1997)+ However, the efficiency of incorporation of NTPaS and dNTPaS by the two polymerases differed+ It was found that a 60% replacement of an NTP by the corresponding dNTPaS in the transcription mixture gave a reasonably detectable signal upon iodine cleavage, and that a similar band intensity could be reached with a 20% replacement by an NTPaS (data not shown)+ These substitution levels were chosen for the interference analysis+ A critical part of the analysis is the separation of the charged from the uncharged tRNAs+ Efforts to separate the two species by gel electrophoresis at low pH (Varshney et al+, 1991) were not satisfactory in our case, nor were the attempts to use affinity electrophoresis after thiolation of the charged tRNA (Igloi, 1992)+ However, specific biotinylation of the charged tRNA and separation from the uncharged tRNA on Streptavidin columns gave reproducible results (Pütz et al+, 1997)+ The ratios of the corresponding bands in the iodine cleavage pattern for each phosphorothioate or 29-deoxy-phosphorothioate position in the aminoacylated and nonaminoacylated fraction were calculated according to F aa ϭ I aa ϫ 100%/(I aa ϩ I na ) and F na ϭ I na ϫ 100%/(I aa ϩ I na )…”

Section: Resultsmentioning

confidence: 99%

Determination of 2′-hydroxyl and phosphate groups important for aminoacylation of Escherichia coli tRNAAsp: A nucleotide analogue interference study

Vörtler

Fedorova

Persson

et al. 1998

RNA

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“…Comparison of RNA-RNA interactions in U2-and U12-dependent spliceosomal splicing+ A: Diagram of the interactions between the pre-mRNA and U1, U2, and U6 snRNAs+ Shown are the U2-branch site interaction, the U1 and U6-59 splice site interactions and the Helix Ia and Ib interactions between U2 and U6 snRNAs+ Also shown is the U6 intramolecular stem-loop structure that immediately follows Helix Ib+ B: Diagram of the analogous interactions between the pre-mRNA and U11, U12, and U6atac snRNAs+ Shown are the U12-branch site interaction, the U11 and U6atac-59 splice site interactions and the U12-U6atac interactions and the U6atac stem-loop structure+ Also shown in the shaded boxes are the mutations used in the in vivo mutational suppression assay+ The P120 CC5/6GG mutation shown in the middle of the upper box inactivates U12-dependent splicing+ The U6atac GG14/15CC mutation restores splicing at the mutant 59 splice site and the U11 GG6/7CC mutation enhances the level of suppression+ functionally active snRNA when tested in vivo (Shukla & Padgett, 1999)+ In addition to the indirect evidence of the importance of the U6 snRNA intramolecular stem-loop provided by phylogenetic conservation, experimental support has also been provided by several studies+ Genetic suppression experiments in yeast have shown that formation of the stem-loop structure is required for splicing (Fortner et al+, 1994;McPheeters, 1996)+ Similar experiments in mammalian systems also showed a requirement for this structure (Wolff & Bindereif, 1993;Sun & Manley, 1995+ In an extensive set of experiments, Sun and Manley (1997) used an in vivo approach to show that U6 snRNA function was maintained as long as the base pairing pattern and a critical U residue in the bulge were conserved+ Other structural modifications were also compatible with function including pairing of the bulged U residue and extension of the helix by an additional base pair+ These results are quite surprising in light of the very high conservation of this region over more than a billion years of evolution+ Results from in vitro modification studies of residues within the U6 intramolecular stem-loop provide additional support for its role in splicing+ In both yeast (Fabrizio & Abelson, 1992) and nematode (Yu et al+, 1995) in vitro splicing systems, phosphorothioate modification of certain phosphodiester bonds blocks splicing+ Such a block could be due to disruption of either RNAprotein interactions or interactions with functional chemical groups such as metal ions required for catalysis or the maintenance of a catalytically active structure (Eckstein, 1985)+ A recent analysis of one of these positions in yeast U6 snRNA has identified a critical metal-ion binding site in the bulge region (Yean et al+, 2000)+ These and other results have led to speculation that this element of U6 snRNA functions at or near the catalytic center of the spliceosome (reviewed in Nilsen, 1998;Collins & Guthrie, 2000)+ Because our earlier experience with substituting the plant U6atac snRNA stem-loop into human U6atac suggested that there was significant sequence flexibility ...…”

Section: Introductionmentioning

confidence: 99%

The intramolecular stem-loop structure of U6 snRNA can functionally replace the U6atac snRNA stem-loop

Shukla

Padgett

2001

RNA

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The U6 spliceosomal snRNA forms an intramolecular stem-loop structure during spliceosome assembly that is required for splicing and is proposed to be at or near the catalytic center of the spliceosome. U6atac snRNA, the analog of U6 snRNA used in the U12-dependent splicing of the minor class of spliceosomal introns, contains a similar stem-loop whose structure but not sequence is conserved between humans and plants. To determine if the U6 and U6atac stem-loops are functionally analogous, the stem-loops from human and budding yeast U6 snRNAs were substituted for the U6atac snRNA structure and tested in an in vivo genetic suppression assay. Both chimeric U6/U6atac snRNA constructs were active for splicing in vivo. In contrast, several mutations of the native U6atac stem-loop that either delete putatively unpaired residues or disrupt the putative stem regions were inactive for splicing. Compensatory mutations that are expected to restore base pairing within the stem regions restored splicing activity. However, other mutants that retained base pairing potential were inactive, suggesting that functional groups within the stem regions may contribute to function. These results show that the U6atac snRNA stem-loop structure is required for in vivo splicing within the U12-dependent spliceosome and that its role is likely to be similar to that of the U6 snRNA intramolecular stem-loop.

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Nucleoside Phosphorothioates

Cited by 905 publications

References 0 publications

Recognition of Nucleoside Triphosphates during RNA-Catalyzed Primer Extension

Recognition of Nucleoside Triphosphates during RNA-Catalyzed Primer Extension

Determination of 2′-hydroxyl and phosphate groups important for aminoacylation of Escherichia coli tRNAAsp: A nucleotide analogue interference study

The intramolecular stem-loop structure of U6 snRNA can functionally replace the U6atac snRNA stem-loop

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