Oligonucleotide duplexes containing 2′-amino-2′-deoxycytidines: thermal stability and chemical reactivity

Aurup, Helle; Tuschl, Thomas; Benseler, Fritz; Ludwig, János; Eckstein, Fritz

doi:10.1093/nar/22.1.20

Cited by 133 publications

(100 citation statements)

References 27 publications

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“…3 These data suggest that G NH 3 + binds more strongly than G or G NH 2 G NH 2 ) 3.6 mM -1 , respectively. 4 This fit also gives a pK a value of 6.1 ( 0.2 for deprotonation of G NH 3 + in solution (Scheme 3, pK a G N ), the same as the solution pK a of 6.2 determined directly for the 2′-amino group within a dinucleotide (45). This solution pK a and the 19-fold stronger binding of G NH 3 + than G NH 2 predict that G NH 3 + deprotonates 4 The binding constant of G NH 3 + is apparent because G NH 3 + binding is coupled to dissociation of a Mg 2+ ion from site C (see Figure 2); in contrast, a Mg 2+ ion is bound at site C in both the E‚S and E‚S‚GNH 2 complexes at 10 mM Mg 2+ (1), and this is denoted by the subscript 'Mg' in the binding constant for GNH 2 (Scheme 3, K b,Mg G NH 2 ).…”

Section: G Nh 3 + Is At Least 10 4 -Fold Less Reactive Than G In the supporting

confidence: 60%

Protonated 2‘-Aminoguanosine as a Probe of the Electrostatic Environment of the Active Site of the Tetrahymena Group I Ribozyme

1999

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We have probed the electrostatic environment of the active site of the Tetrahymena group I ribozyme (E) using protonated 2′-aminoguanosine (G NH 3 +), in which the 2′-OH of the guanosine nucleophile (G) is replaced by an -NH 3 + group. At low concentrations of divalent metal ion (2 mM Mg 2+ ), G NH 3 + binds at least 200-fold stronger than G or G NH 2 , with a dissociation constant of e1 µM from the ribozyme‚oligonucleotide substrate‚G NH 3 + complex (E‚S‚G NH 3 +). This strong binding suggests that the -NH 3 + group interacts with negatively charged phosphoryl groups within the active site. Increasing the concentration of divalent metal ion weakens the binding of G NH 3 + to E‚S more than 10 2 -fold. The Mn 2+ concentration dependence suggests that M C , the metal ion that interacts with the 2′-moiety of G in the normal reaction, is responsible for this effect. M C and G NH 3 + compete for binding to the active site; this competition could arise from electrostatic repulsion between the positively charged -NH 3 + and M C and, possibly, from their competition for interaction with active site phosphoryl groups. The reactive phosphoryl group of S increases the competition between M C and G NH 3 +, consistent with a network of interactions involving M C that help position the reactive phosphoryl group and the guanosine nucleophile with respect to one another. The chemical step with bound G NH 3 + is at least 10 4 -fold slower than with G or G NH 2 . These results provide additional support for an integral role of M C in catalysis by the Tetrahymena ribozyme, and demonstrate the utility of the -NH 3 + moiety as an electrostatic probe within a structured RNA.An RNA enzyme can utilize a variety of functional groups that differ in charge and polarity for building its active site and interacting with its substrates. The backbone of RNA is composed of negatively charged phosphoryl groups; this results in repulsive interactions, but also creates numerous potential binding sites for metal ions and other positively charged groups (e.g., 2-15 and references cited therein). The 2′-hydroxyl groups and functional groups on the bases of RNA also provide hydrogen bond donors and acceptors that can interact with each other, with metal ions, and with polar groups on bound ligands (e.g., 3, 4, 6, 8, 9, 11-14, 16-21, and references cited therein). The ring moieties of the bases are nonpolar, creating the potential for hydrophobic interactions with each other and with bound ligands (e.g., 19, 21, and references cited therein).The energetic effects of electrostatic interactions between functional groups within an RNA also depend on the ability of the RNA to position these groups and to limit active site rearrangements. The Tetrahymena group I ribozyme makes extensive interactions with its substrates, which has been suggested to allow precise positioning of substrates and catalytic groups within this RNA active site (22-25 and references cited therein). The interactions within the ribozyme core presumably form an interconnecte...

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Section: G Nh 3 + Is At Least 10 4 -Fold Less Reactive Than G In the supporting

confidence: 60%

Protonated 2‘-Aminoguanosine as a Probe of the Electrostatic Environment of the Active Site of the Tetrahymena Group I Ribozyme

1999

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“…The pH of 7.0 was chosen to provide conditions where the 2Ј-amino group is mainly deprotonated. The pK a of the 2Ј-amino group was determined to be 6.2 (25) Cleavage efficiency of all-ribose and 2Ј-modified ptRNA Gly CU variants at pH 7.0 decreased in the order 2Ј-OH Ͼ 2Ј-F Ͼ 2Ј-N Ͼ 2Ј-H ( Fig. 2A).…”

Section: Resultsmentioning

confidence: 96%

Catalysis by RNase P RNA

Persson

Cuzic

Hartmann

2003

Journal of Biological Chemistry

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Metal ions are essential cofactors for precursor tRNA (ptRNA) processing by bacterial RNase P. The ribose 2-OH at nucleotide (nt) ؊1 of ptRNAs is known to contribute to positioning of catalytic Me 2؉ . To investigate the catalytic process, we used ptRNAs with single 2-deoxy (2-H), 2-amino (2-N), or 2-fluoro (2-F) modifications at the cleavage site (nt ؊1). 2 modifications had small (2.4 -7.7-fold) effects on ptRNA binding to E. coli RNase P RNA in the ground state, decreasing substrate affinity in the order 2-OH > 2-F > 2-N > 2-H. Effects on the rate of the chemical step (about 10-fold for 2-F, almost 150-fold for 2-H and 2-N) were much stronger, and, except for the 2-N modification, resembled strikingly those observed in the Tetrahymena ribozyme-catalyzed reaction at corresponding position. Mn 2؉ rescued cleavage of the 2-N but also the 2-H-modified ptRNA, arguing against a direct metal ion coordination at this location. Miscleavage between nt ؊1 and ؊2 was observed for the 2-N-ptRNA at low pH (further influenced by the base identities at nt ؊1 and ؉73), suggesting repulsion of a catalytic metal ion due to protonation of the amino group. Effects caused by the 2-N modification at nt ؊1 of the substrate allowed us to substantiate a mechanistic difference in phosphodiester hydrolysis catalyzed by Escherichia coli RNase P RNA and the Tetrahymena ribozyme: a metal ion binds next to the 2 substituent at nt ؊1 in the reaction catalyzed by RNase P RNA, but not at the corresponding location in the Tetrahymena ribozyme reaction.Endonucleolytic 5Ј maturation of tRNA primary transcripts is catalyzed by the ribonucleoprotein enzyme ribonuclease P (RNase P) 1 in all three domains of life (Archaea, Bacteria, and Eukarya) as well as in mitochondria and chloroplasts (1-3). Bacterial RNase P enzymes are composed of a catalytic RNA subunit (4), ϳ400 nucleotides (nt) in length, and a single small protein of typically 120 amino acids (5). The only known exception may be the hyperthermophilic bacterium Aquifex aeolicus.Neither genes for RNase P RNA (rnpB) or protein (rnpA) subunits could be identified in the sequenced genome, nor have biochemical studies provided evidence for the presence of a bacterial-like RNase P activity (6).Processing of ptRNAs by RNase P results in cleavage products with 3Ј-OH and 5Ј-phosphate termini. Studies with the model RNase P RNA ribozymes from Escherichia coli and Bacillus subtilis have indicated that at least two metal ions (the actual number influenced by the nature and concentration of monovalent cations present) play a specific role in productive enzyme-substrate complex formation and cleavage chemistry (7-14). A solvent hydroxide or activated water molecule is assumed to be positioned by a metal ion for nucleophilic attack in an S N 2 in-line displacement mechanism (7, 15). A 2Ј-deoxyribose substitution at the RNase P cleavage site was previously reported to affect binding of catalytically important Mg 2ϩ and to substantially reduce the rate of catalysis by E. coli RNase P RNA (7, 16 -18). Recent ...

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“…Following binding of G(2 ′ NH 2 ) and G(3 ′ NH 2 ) to the ribozyme The 2 ′ -NH 2 and 3 ′ -NH 2 groups of G(2 ′ N) and G(3 ′ N), respectively, can ionize to form the corresponding −NH 3 + species (Aurup et al 1994;Dai et al 2007). To conduct metal ion rescue experiments with G(2 ′ NH 2 ) and G(3 ′ NH 2 ) (and not G(2 ′ NH 3 + ) and G(3 ′ NH 3 + )), we established conditions under which we were able to obtain affinities for the -NH 2 forms of G(2 ′ N) and G(3 ′ N) to (E•S) O , (E•S) C , (E•P) C , as described below.…”

Section: Measurement Of G Analog Affinities To (E•p) Cmentioning

confidence: 99%

An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions

Sengupta

Schie

Giambaşu

et al. 2015

RNA

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Biological catalysis hinges on the precise structural integrity of an active site that binds and transforms its substrates and meeting this requirement presents a unique challenge for RNA enzymes. Functional RNAs, including ribozymes, fold into their active conformations within rugged energy landscapes that often contain misfolded conformers. Here we uncover and characterize one such "off-pathway" species within an active site after overall folding of the ribozyme is complete. The Tetrahymena group I ribozyme (E) catalyzes cleavage of an oligonucleotide substrate (S) by an exogenous guanosine (G) cofactor. We tested whether specific catalytic interactions with G are present in the preceding E•S•G and E•G ground-state complexes. We monitored interactions with G via the effects of 2 ′ -and 3 ′ -deoxy (-H) and −amino (-NH 2 ) substitutions on G binding. These and prior results reveal that G is bound in an inactive configuration within E•G, with the nucleophilic 3 ′ -OH making a nonproductive interaction with an active site metal ion termed M A and with the adjacent 2 ′ -OH making no interaction. Upon S binding, a rearrangement occurs that allows both -OH groups to contact a different active site metal ion, termed M C , to make what are likely to be their catalytic interactions. The reactive phosphoryl group on S promotes this change, presumably by repositioning the metal ions with respect to G. This conformational transition demonstrates local rearrangements within an otherwise folded RNA, underscoring RNA's difficulty in specifying a unique conformation and highlighting Nature's potential to use local transitions of RNA in complex function.

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Oligonucleotide duplexes containing 2′-amino-2′-deoxycytidines: thermal stability and chemical reactivity

Cited by 133 publications

References 27 publications

Protonated 2‘-Aminoguanosine as a Probe of the Electrostatic Environment of the Active Site of the Tetrahymena Group I Ribozyme

Protonated 2‘-Aminoguanosine as a Probe of the Electrostatic Environment of the Active Site of the Tetrahymena Group I Ribozyme

Catalysis by RNase P RNA

An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions

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