correspondence of functional and microscopic pka values in a ribozyme the ph dependence of

Correspondence of functional and microscopic pKa values in a ribozyme

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The pH Dependence of Hairpin Ribozyme Catalysis Reflects Ionization of an Active Site Adenine*

Joseph W. Cottrell1, Lincoln G. Scott2, and Martha J. Fedor1

1From the Department of Chemical Physiology, the Department of Molecular Biology and The Skaggs

Institute for Chemical Biology The Scripps Research Institute, La Jolla, CA 92037

2Cassia, LLC, San Diego, CA 92109

*Running title: Correspondence of functional and microscopic pKa values in a ribozyme

To whom correspondence should be addressed: Martha J. Fedor, Department of Chemical Physiology, Department of Molecular Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA, USA, Tel.: (858) 784-2770; Fax: (858) 784-2779; E-mail: [email protected] Keywords: RNA catalysis; ribozyme; fluorescence Background: The hairpin ribozyme is a prototype of RNA enzymes that mediate catalysis without divalent cation cofactors. Results: Self-cleavage activity decreases as the fraction of the protonated form of an active site adenosine increases. Conclusion: The neutral, unprotonated form of adenosine interacts with the nucleophilic- or leaving-group oxygen to facilitate catalysis. Significance: Learning how RNA functional groups participate in catalysis is crucial for understanding RNA-mediated processes in biology. SUMMARY

Understanding how self-cleaving ribozymes mediate catalysis is crucial in light of compelling evidence that human and bacterial gene expression can be regulated through RNA self-cleavage. The hairpin ribozyme catalyzes reversible phosphodiester bond cleavage through a mechanism that does not require divalent metal cations. Previous structural and biochemical evidence implicated the amidine group of an active site adenosine, A38, in a pH dependent step in catalysis. We developed a way to determine microscopic pKa values in active ribozymes based on the pH-dependent fluorescence of 8-azaadenosine. We compared the microscopic pKa for 8azaA38 ionization to the apparent pKa for the self-cleavage reaction in a fully functional hairpin ribozyme with a unique 8-azaadenosine at position 38.

Microscopic and apparent pKa values were virtually the same, evidence that A38 protonation accounts for the decrease in catalytic activity with decreasing pH. These results implicate the neutral, unprotonated form of A38 in a transition state that involves formation of the 5'-oxygen-phosphorus bond.

Hairpin ribozymes (Hp Rzs) belong to one of several families of small self-cleaving RNAs that serve as useful models of RNA catalysis because they are relatively simple and amenable to chemogenetic analyses (1). The Hp Rz remains functional in the absence of divalent metals, relying exclusively on nucleotide functional groups for catalytic chemistry (2-5). High resolution structures of the Hp Rz bound to transition state mimics show two active site purines, G8 and A38, positioned in a similar manner to the two histidines in RNase A that mediate general acid base catalysis of the same reaction (6-8) (Fig. 1A), leading to the proposal that the hairpin ribozyme uses the same concerted general acid base mechanism (9) (Fig. 1B). A38(N1) is near the 5’-oxygen of the reactive phosphodiester, and A38(N6) lies within hydrogen bonding distance of the pro-RP non-bridging oxygen. Exogenous nucleobase rescue experiments confirmed that the amidine group of A38 interacts with the transition state but its precise role remained unclear (10). In a general acid base model, the cationic, protonated form of A38 would act as a general acid during cleavage


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to donate a proton to the 5’-oxygen leaving group as the 5'-oxygen phosphorus bond breaks. During the reverse ligation reaction, unprotonated A38 would accept a proton to activate the nucleophilic 5' oxygen for attack on phosphorus as the 5' oxygen phosphorus bond forms. Cleavage and ligation rate constants both increase with increasing pH, and exhibit the same apparent pKa values near 6.5 (3), consistent with the law of microscopic reversibility (11). The pH dependence of reaction kinetics can provide information about the identity of general acid base catalyst when an apparent pKa value clearly corresponds with the microscopic pKa value for a particular active site functional group. However, no RNA functional groups exhibit protonation equilibria near pH 6.5, at least as free nucleotides in solution (12), although nucleotide functional groups are well known to shift in the context of specific RNA structures (13-15). In the case of the Hp Rz, however, no candidate for a general acid base catalyst could be identified from comparison of apparent pKa values obtained from pH-rate profiles and microscopic pKa values for protonation of particular RNA functional groups.

Considerable evidence implicates A38, but not G8, in the pH dependent step of the reaction pathway. Substitution of A38 with an abasic linkage or with N1-deazaadenosine reduces activity 104-fold and 107-fold, respectively (10,16). In Hp Rzs with an abasic linkage in place of A38, pKa,app values shift from 6 to 9 (10). Substitutions of A38 with nucleotide analogs that differ in intrinsic acidity have corresponding effects on the pH dependence of Hp Rz cleavage and ligation activity (10,17). Activity was restored to abasic A38 ribozymes with certain exogenous nucleobases that share the amidine group of adenine, and the pH dependence of chemical rescue reactions correlated with the intrinsic basicity of the rescuing nucleobase. In contrast, Hp Rzs with an abasic linkage in place of G8 exhibit an 850-fold loss of self-cleavage activity, but the apparent pKa for the reduced activity remains near 6 (18). Our recent evidence that the microscopic pKa values are above 9 for 8azaG ionization in 8azaGHp Rzs and do not correlate with the pKa,app values below 7 obtained from 8azaGHp Rz pH-rate profiles also argues that the pH-dependent step in the hairpin ribozyme reaction reflects the protonation state of A38 rather than G8 (19).

The general problem of kinetic equivalence limits the ability to distinguish among alternative catalytic mechanisms using kinetic data alone (12).

In the case of the Hp Rz, the same increase in activity with increasing pH could be observed if donation of a proton to the 5' oxygen by the protonated form of adenosine and withdrawal of a proton from the 2' oxygen by hydroxide ion occurred in the transition state or if the reaction involved unprotonated adenosine alone (10,20).

Molecular dynamics simulations support a model in which the protonated form of A38 participates in catalysis as a general acid (21) and a model in which A38 both accepts a proton from the nucleophilic 2'-oxygen and donates a proton to the departing 5'-oxygen leaving group during cleavage (22). A38 might also facilitate catalysis through mechanisms other than general acid base catalysis (1). Hydrogen bonding interactions between A38(N1) with the 5' oxygen and between A38(N6) and the pro-RP oxygen might facilitate catalysis by positioning and orientating reactive groups in the trigonal bipyramidal geometry needed for an SN2(P)-type in-line attack mechanism. Molecular dynamics simulations also support a model in which active site interactions provide electrostatic stabilization of the electronegative transition state without direct participation of RNA functional groups in proton transfer (23). Finally, the decrease in Hp Rz activity at low pH might just reflect the destabilization that is characteristic of all RNA structures at pH extremes that disrupt hydrogen bonds and might not be related to the catalytic mechanism at all. Information about the protonation state of A38(N1) in a functional ribozyme would help to distinguish among these alternative possibilities.

We set out to gain a better understanding of how RNA functional groups can participate in catalysis and to distinguish among alternative roles for A38 in the Hp Rz mechanism by determining the protonation state of A38(N1) directly in a functional ribozyme. 8-azaadenosine (8azaA) is an adenosine analog that exhibits high fluorescence emission intensity when the N1 position is unprotonated whereas emission intensity is low when N1 is protonated (24) (Fig. 2A). 8-azaadenine differs from adenine only in having nitrogen in place of carbon at position 8 and maintains the Watson-Crick hydrogen bonding face of adenine (Fig. 2B). Moreover, Hp Rzs with 8azaA in place of A38 retain full activity (25). We developed a novel method to place a single, unique 8azaA at position 38 by using 8-azaadenosine 5’-monophosphate


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(8azaAMP) to prime in vitro transcription of a ribozyme fragment with A38 at the 5' end (Fig. 3). Ligation of the 5'-8azaA38 RNA to an unmodified transcript containing the remaining ribozyme sequence produced a complete, functional ribozyme.

Microscopic pKa values determined from pH-fluorescence profiles were virtually the same as pKa,app values calculated from pH-rate profiles for the 8azaA Hp Rz. Our results link A38(N1) protonation directly to the decrease in Hp Rz activity with decreasing pH. These results contradict the idea that the cationic protonated form of A38 is essential for catalysis. Instead, they point to a view of the Hp Rz transition state in which the neutral, unprotonated form of A38 facilitates formation of the 5' oxygen-phosphorus bond. EXPERIMENTAL PROCEDURES Preparation of 8azaAMP – Step 1 (Fig. 3A-C): to a 500 mL round bottom flask, D-ribose (300 mg, 2.0 mmol) and 8azaA (273 mg, 2.0 mmol, MP Biomedicals) were dissolved in 200 mL of pH 7.5 water containing: 0.008 mM dATP, 0.05 mM kanamycin, 0.15 mM ampicillin, 0.4 mM dAMP, 20 mM MgCl2, 20 mM dithiothreitol, 50 mM potassium phosphate, 100 mM creatine phosphate. The synthesis was started with the addition of 10 units of ribokinase (rbsK, EC 2.7.1.15), 15 units of 5-phospho-D-ribosyl-α-1-pyrophosphate synthetase (prsA, EC 2.7.6.1), 20 units of adenine phosphoribosyltransferase (aprT, EC 2.4.2.7), 25 units of inorganic pyrophosphatase (hppA, EC 3.6.1.1), 65 units of adenylate kinase (plsA, EC 2.7.4.3), and 125 units of creatine phosphokinase (ckmT, EC 2.7.3.2). After 48 hours the synthesis of 8-azaadenosine-5'-triphosphate (8azaATP) appeared complete by analytical HPLC. The reaction was brought to 0.5 M ammonium bicarbonate and pH 9.5 with addition of ammonium hydroxide, and purified by boronate-affinity chromatography. Step 2 (Fig. 3D): 8azaATP (1.2 mmol, 60% isolated yield) and D-ribose (180 mg, 1.2 mmol) was added to a 250 mL round bottom flask and dissolved in 120 mL of pH 7.5 water containing: 0.05 mM kanamycin, 0.15 mM ampicillin, 20 mM MgCl2. The stoichiometric dephosphorylation of 8azaATP to 8azaAMP was started with the addition of 10 units of rbsK, 15 units of prsA, and 65 units of plsA. After 48 hours the synthesis of 8azaAMP appeared complete by analytical HPLC. The reaction was again brought to 0.5 M ammonium bicarbonate (pH 9.5), and

purified by boronate-affinity chromatography (0.5 mmol, 40% isolated yield). Data: 1H-NMR (600 MHz, D2O) d (mult.) 8.21 (s, H-8), 6.20 (d, H-1'), 4.97 (dd, H-2'), 4.54 (dd, H-3'), 4.14 (ddd, H-4'), 3.91 (dd, H-5'). .HRMS (ESI-TOF+) m/z calcd [M+H]+ 349.0662, found 349.0656; HRMS (ESI-TOF-) m/z calcd (M-H]- 347.0505, found 347.0508.

RNA preparation– RNAs were prepared by bacteriophage T7 RNA polymerase transcription of linearized plasmid templates or of partially duplex synthetic oligonucleotide templates, or by chemical synthesis (Dharmacon) as described previously (26,27). Plasmid templates encoding Hp Rz RNAs were prepared using conventional molecular cloning and PCR mutagenesis methods, as described (26,27).

The self-cleaving Hp Rz variants used for these experiments assemble in the context of a four-way helical junction and have either three or eight base pairs in the intermolecular H1 helix that forms between the 5' and 3' cleavage product RNAs (Fig. 4). Self-cleavage of Hp Rzs with 4-way helical junctions and eight base pairs in H1 forms a stable 8azaA5'R.P complex with an internal equilibrium that favors the ligated ribozyme relative to 5'R and 3'P cleavage products under the conditions used for fluorescence experiments (26). In Hp Rzs with just three base pairs in H1, self-cleavage products dissociate rapidly, preventing re-ligation of bound products that could complicate interpretation of cleavage rate measurements (26,28).

8azaAR (Fig. 4, blue) was transcribed in reactions with 4 mM 8azaAMP and 4 mM each NTP. 8azaAR was ligated to the ∆A∆BHp48 RNA (Fig. 4, green) to generate the complete 8azaAHp Rz RNA (29). Before ligation, 40 µM each ∆A∆BHp48 RNA and 8azaAR RNA were heated to 85˚ C and cooled to 15˚ C in the presence of 10 mM MgCl2. Ligation was carried out at 15˚ C for 18 hours in reactions with 1 mM ATP, and 2 units/µL of T4 RNA ligase 1. Unmodified ribozymes prepared through ligation exhibited virtually the same self-cleavage kinetics as ribozymes prepared as single transcripts. 8azaAHpm Rz RNAs were prepared in the same way as 8azaAHp Rz RNAs but have an inactivating G+1A mutation. Self-cleavage kinetics–Self-cleavage rates were determined using ligation-chase assays with 32pCp-labeled 3’ product RNAs and 5' ribozyme


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RNAs, as described (26). Assays were performed at 25° C in 50 mM buffers at various pH values (PIPPS pH 3-4.6, MES pH 4.7-7, HEPES pH 7-8) with 10 mM MgCl2 and 0.1 mM EDTA. Reported values are the mean of two or more independent experiments. Reported errors are standard deviations. Apparent pKa values were determined by fitting self-cleavage rates to Equation 1 and errors were obtained from least squares analyses of the fits.

(1)

Fluorescence measurements–Fluorescence was measured using a SpectraMax M2e plate reader (Molecular Devices). 10 µM 8azaAMP or 15 µM 8azaAHp Rz RNA were prepared in 50 mM buffer (glycine pH 1 – 2.4, PIPPS pH 2.4 – 4.6, MES pH 4.7 – 7, HEPES pH 7 – 8, TAPS pH 8 – 9), 10 mM MgCl2 and 0.1 mM EDTA. RNAs were annealed by heating to 85˚ C for 1 minute and slowly cooled to 25˚ C, with addition of MgCl2 at 60˚ C. A 20 µL aliquot was added to 384-well black microplates (Greiner). During excitation at 290 nm, emission spectra were recorded between 310 nm and 450 nm. Emission intensities for RNAs with adenosine rather than 8-azaadenosine at position 38 were subtracted from emission spectra for 8azaA RNAs to correct for non-specific fluorescence and UV absorption. pKa values were determined by fitting integrated fluorescence intensities to Equation 2,

(2)

where FB and FBH are the normalized fluorescence emission intensities of unprotonated and protonated 8azaA, respectively and Ka is the acid dissociation constant for protonation of 8azaA in 8azaAMP and in Hp Rz RNAs. Reported errors were obtained from least squares analyses of the fits. RESULTS

Synthesis of 8azaAMP–8azaAMP was prepared using an in vitro enzymatic synthesis method beginning with the C-5-phosphorylation of ribose by ribokinase (rbsK), to give ribose-5-phosphate (R5P) that is further phosphorylated at the C-1 position by 5-phospho-D-ribosyl-α-1-

pyrophosphate synthase (prsA) forming 5-phospho-D-ribosyl-α-1-pyrophosphate (PRPP) (Fig. 3). Adenine phosphoribosyltransferase (aprT) efficiently coupled 8-azaadenine to PRPP, forming 8azaAMP. The monophosphate was subsequently converted to 8azaATP by sequential phosphorylation of adenylate kinase (plsA) and creatine phosphokinase (ckmT) in good isolated yield. Due to the lack of a catalytically active 8azaATP pyrophosphatase, the second step of the synthesis involved essentially the reverse reaction, in order to discharge the 8azaATP to 8azaAMP. Key features of this synthesis include the use of dATP as the phosphate donor, and dATP being easily separated from the ribonucleotide analog by boronate affinity chromatography.

A hairpin ribozyme with 8azaA in place of an essential active site adenosine–8azaAHp Rz is a hairpin ribozyme sequence variant that was designed to allow substitution of a single 8-azaadenosine for the critical adenosine at position 38 in the active site (Fig. 4). In our previous studies of 8-azaguanosine-substituted ribozymes, 8azaGHp Rz RNAs were prepared by transcription of a template with a unique cytosine in reactions with 8azaGTP in place of GTP and then ligating the 8azaG transcript to an unmodified transcript to give the complete ribozyme. We initially attempted to prepare a ribozyme with a unique 8azaA38 in the same way. However, ligation of three RNA fragments was required to make a complete Hp Rz RNA with a single 8azaA38 due to the presence of other important adenosines close to A38. Three-part ligations did not produce 8azaAHp Rz RNAs in sufficient yields to compensate for the lower quantum yield of 8azaA relative to 8azaG (Fig. 2). Therefore, we developed a new approach using 8azaAMP to place a unique 8azaA as the first nucleotide of an in vitro transcript (Fig. 4, 8azaAR, blue). Bacteriophage T7 RNA polymerase greatly prefers guanosine as the 5' terminal nucleotide (30). To improve transcription efficiency, the uridine at position 39 in the wild type ribozyme sequence was changed to guanosine. This change was not expected to affect self-cleavage activity (31) and increased transcription yields more than 20-fold.

Optimal transcription conditions were identified in trials with different NTP, AMP and 8azaAMP concentrations. The efficiency of 8azaAMP and AMP incorporation was monitored


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through reduction in [ϒ-32P]ATP labeling. Overall transcription yields were monitored in parallel reactions with [α-32P]ATP. Optimized reactions with 4 mM each 8azaAMP, ATP, GTP, CTP, and UTP produced 32 transcripts per template. The transcription efficiency of reactions with 8azaAMP was somewhat lower than reactions with AMP, which produced 44 transcripts per template under the same conditions. These yields are comparable to yields obtained in conventional transcription reactions that include only NTPs (30).

5'-8azaAMP-terminated 8azaAR was ligated to a second unmodified transcript (Fig. 4, green) to form the complete ribozyme, giving 15% and 60% yields of 8azaAHp Rz and Hp Rz RNAs, respectively. 5' ends of transcripts that initiate with ATP would not undergo ligation. The 4-fold lower yields of 8azaAHp Rz RNAs relative to Hp Rz RNAs prepared through ligation of transcripts primed with AMP might reflect a larger fraction of transcripts with 5' terminal ATP that do not undergo ligation, which might be higher in transcription reactions with 8azaAMP instead of AMP. Ligation efficiency might also be lower if 8azaA transcripts are poor substrates for T4 RNA ligase 1. As expected from early mutagenesis studies (31), the U39G Hp Rz exhibited virtually the same cleavage rate constants and pH dependence as HP43, the parental Hp Rz characterized previously (26).

The pH dependence of 8azaAHp Rz self-cleavage activity–8azaAHp Rzs exhibited approximately 10-fold lower activity than the same Hp Rz sequence with unmodified adenosine at position 38 (Fig. 5A). Both 8azaA-substituted and unmodified ribozymes exhibited pH-dependent self-cleavage kinetics that fit well to an equation that includes a variable for a single ionizable group. An apparent pKa value of 6 was determined from the pH dependence of self-cleavage kinetics of this Hp Rz sequence variant with unmodified adenosine at position 38. This value is similar to the pKa,app values previously determined from the pH dependence of self-cleavage and ligation kinetics for other Hp Rz variants (10,18). An apparent pKa value of 4 was determined from the pH dependence of 8azaAHp Rz self-cleavage kinetics (Fig. 5A, black), a shift of 2 units in the acidic direction relative to the pKa,app of unmodified Hp Rzs (Fig. 5A, green). This shift is similar to the acidic shift in microscopic pKa values from 3.52 to 2.2 for N1 protonation in adenosine and 8-azaadenosine, respectively (24,32).

Microscopic pKa for N1 protonation of 8azaA38 in the Hp Rz active site. pH-fluorescence profiles were measured for 8azaAMP, 8azaAHp, and 8azaAHpm RNAs under the same conditions used for measuring self-cleavage kinetics. 8azaAHp and 8azaAHpm RNAs exhibited a 40-fold lower quantum yield than 8azaAMP. RNAs with 8-azaguanosine substitutions exhibited a similar level of fluorescence quenching relative to 8azaGTP (19,33). Quenching of 8-azapurine fluorescence in RNAs is likely attributable to orbital overlap with flanking nucleobases as characterized previously for 2-aminopurine in B-form helices (34-36).

Microscopic pKa values for N1 protonation of 8azaA in 8azaAMP, 8azaAHp Rz and 8azaAHpm Rz were calculated from the pH-dependence of fluorescence emission intensities (Fig. 5B). The pKa value of 2.5 that was determined for 8azaAMP ionization is slightly more basic than the pKa value of 2.2 reported for 8-azaadenosine (24). A similar small basic shift is observed for other nucleotides relative to nucleosides, a shift that probably reflects the influence of the negatively charged phosphate (37).

RNA was not sufficiently soluble below pH 4.2 to permit measurements of fluorescence emission spectra. To learn how uncertainty in the determination of the fluorescence emission intensity of protonated 8azaA in RNA at low pH affected pKa calculations, we compared microscopic pKa values that were calculated by fixing or not fixing the minimum fluorescence emission intensity of fully protonated 8azaA at the value of 0.23 that we obtained from the pH dependence of 8azaAMP fluorescence across the full pH range. pKa values obtained from both calculation methods agreed within 0.2 pH units, with values calculated without fixing the minimum fluorescence emission intensity being slightly more basic. The reported fits and pKa values in Fig. 5B were obtained with the fixed value.

The microscopic pKa of 4.6 (Fig. 5B, green) for ionization of 8azaA38 in 8azaAHp Rz RNA was shifted by 2.1 units in the basic direction relative to the pKa value of 2.5 obtained for 8azaAMP (Fig. 5B, black). Thus, some feature of the active site environment facilitates N1 protonation relative to the behavior of 8azaA in solution. 8azaA38 exhibited a similar, but


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slightly more basic pKa shift of 2.5 units in 8azaAHpm Rz RNA (Fig. 5B, red), evidence that the mutationally inactivated ribozyme retains the active site features that are responsible for the basic shift. These microscopic pKa values for 8azaA protonation in ribozyme RNAs are experimentally indistinguishable from the apparent pKa value of 4.0 measured for the pH dependent step in the 8azaAHp Rz self-cleavage reaction.

DISCUSSION The importance of nucleobase protonation

states in RNA structure and function has been recognized for some time (13,14,38-51). Nucleobase ionization properties are particularly important in catalytic RNAs where nucleobases are thought to participate directly in proton transfer steps of catalytic chemistry (1). High resolution structures of Hp Rz complexes with transition state mimics place G8 and A38 near the reactive phosphate, positioned like the two histidines in RNase A that mediate general acid base catalysis of the same reaction (6-9) (Fig. 1A), making the protonation state of these nucleobases the focus of considerable interest.

NMR with isotopically labeled RNAs and, more recently, Raman crystallography have been used to measure microscopic pKa values for protonation of N3 of pyrimidines and N1 of purines in catalytic RNAs (42,47,48,51). NMR analyses of A38 protonation in an isolated loop B domain of the hairpin ribozyme gave a pKa value of 4.89 (40). Notably, the acid dissociation constant determined for N1 of A38 in loop B was intermediate between the values measured for other adenosines in the same RNA, evidence that adenosines in a variety of structural contexts exhibit different pKa values than adenosine in solution. Analysis of A38 protonation in a ribozyme inactivated by methylation of the nucleophilic 2'-oxygen using Raman crystallography revealed a pKa value of 5.46 (48). These values are somewhat lower than the pKa,app values near 6 determined from the pH dependence of self-cleavage activity (10,18,19). However, the use of ribozyme fragments or ribozymes with inactivating modifications in these studies complicates the interpretation of these results in terms of nucleobase ionization equilibria in functional ribozymes. 8-Azapurines retain the Watson-Crick hydrogen bonding face of their natural counterparts and differ only slightly in

intrinsic acidity at the N1 position (24) so ribozymes with 8-azaguanosine or 8-azaadenosine substituted for their natural analogs in hairpin ribozymes remain functional (19,25,52).

We report the first application of 8-azaadenosine fluorescence to analyze a catalytically essential adenosine in a functional ribozyme. Application of this method required us to prepare 8azaAHp Rz RNA with 8-azaadenosine uniquely substituted for a single adenosine at position 38 in sufficient yields to give quantifiable emission spectra despite the low quantum yield of 8-azaadenosine. This was accomplished using a novel method for site-specific insertion of a modified nucleotide that involved ligation of two transcripts, one of which was primed with the 5'-monophosphate form of the modified nucleoside. T7 RNA polymerase transcription reactions primed with 8azaAMP gave relatively poor yields. However, reduced yields of 5'-monophosphate-terminated transcripts were balanced by the specificity of 8azaA incorporation that resulted from discrimination against 5'-triphosphate-terminated transcripts during the subsequence ligation step that was used to prepare the complete ribozyme.

The activity of the hairpin ribozyme with an 8azaA38 substitution was reduced about 10-fold relative to an unmodified ribozyme. The reason for the moderate reduction in activity is unclear because the single N8 for C8 change in 8azaA does not affect any interactions that are evident in high resolution structures (6-8). However, deletion of A38 by substitution with an abasic linkage reduces Hp Rz self-cleavage activity by 14,000-fold (10), so 8azaA provides almost the same contribution to catalysis as its natural counterpart. The 8azaAHp Rz exhibited the same increase in self-cleavage activity with increasing pH that is characteristic of unmodified ribozymes (10,18,19).

The apparent pKa value determined from the pH dependence of 8azaHp Rz self-cleavage kinetics was shifted about 2 units in the acidic direction with respect to the apparent pKa,app value of 6 determined for the unmodified Hp Rz. These shifts correspond to the differences in BrØnsted acid strength between 8-azaadenosine, with a pKa value of 2.2, and adenosine, with a pKa of 3.52 for N1 protonation (24,32). Hairpin ribozymes with isoguanine in place of adenosine at position 38 showed a similar correspondence


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between the BrØnsted acid strength of isoguanosine (53) and the pH dependence of reaction kinetics (10). Given the difference of 1.32 units in BrØnsted acid strength of 8azaA and adenosine (24,32), the pKa value of 4.6 that we determined for protonation of 8azaA38 in the Hp Rz active site corresponds with a pKa of 5.92 for protonation of A38 in an unmodified ribozyme. This microscopic pKa value is virtually identical to the apparent pKa value of 6 determined from the pH dependence of self- cleavage activity of the unmodified ribozyme. These strong correlations support the conclusion that the pH-dependent step in the hairpin ribozyme reaction pathway directly involves A38.

The molecular basis of the shift of nucleobase ionization constants in the active site of the ribozyme relative to their values in solution is not yet clear. High-resolution structures of the HP Rz active site reveal stacking interactions above and below A38 and hydrogen bonding interactions with virtually every functional group of A38 (6-8) that might influence ionization equilibria. Most of these interactions are located within loop B. This might account for the similarity in microscopic pKa values between the functional Hp Rz and Hpm Rz, which is inactivated by a G+1A mutation. A tertiary interaction between G+1 in loop A and C25 in loop B is critical for interdomain docking (54,55). Although no bound metal cations were located near A38 in X-ray structures of transition state mimics, cations are required to stabilize the functional ribozyme structure (2-5,56) and nonspecific electrostatic interactions might also influence A38 ionization.

The close correspondence between apparent pKa values for fluorescent and unmodified ribozymes and microscopic pKa values for ionization of N1 in 8-azaadenosine and by extension adenosine (24,57), shows that the pH dependent step in hairpin ribozyme depends directly on the protonation state of A38(N1). The neutral unprotonated form of A38 predominates at pH

values above pH 7 that support optimal activity and A38 protonation is responsible for the decrease in self-cleavage activity with decreasing pH. These results are not consistent with the conventional view of the hairpin ribozyme mechanism that emphasizes the role of the cationic, protonated form of A38 in donating a proton to the departing 5' oxygen during 5'- oxygen-phosphorus bond breaking (Fig. 1B). Instead, these results support a view of the transition state that focuses on the 5'-oxygen- phosphorus bond formation step in which the neutral, unprotonated form of A38 mediates general or specific base catalysis to activate the 5'-oxygen nucleophile for attack on the phosphorus (Fig. 6). Intriguingly, these results contrast with recent evidence that the cationic, protonated form of an active site cytosine predominates at neutral pH where it is thought to serve as a general acid catalysis during self- cleavage of the hepatitis delta virus ribozyme (47). RNA functional groups clearly have the potential to participate in catalysis through a variety of mechanisms.

This study presents a novel method for determining microscopic pKa values for an essential adenosine in the active site of a functional hairpin ribozyme. We applied this method to gain fundamental insight into how RNA functional groups can participate in catalysis. Our results likely have general implications for the mechanisms of other RNA enzymes that function without a requirement for divalent cation cofactors, such as the peptidyl transferase center of the ribosome. The ability to use the pH-dependent fluorescence of a nonperturbing adenosine analog to monitor ionization states might also prove useful for dissecting structure-function relationships in other RNAs.


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Acknowledgments–We thank Julia Viladoms Claverol and Peter Y. Watson for critical reading of the manuscript and JVC for help with figure preparation. FOOTNOTES *This work was supported by NIH grant RO1 GM046422 to MJF. 1To whom correspondence may be addressed: Department of Chemical Physiology, Department of Molecular Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, MB35, La Jolla, CA 92037, USA, Tel.: (858) 784-2770; Fax: (858) 784-2779; E-mail: [email protected] 2Cassia LLC, 3030 Bunker Hill Street, Suite 214, San Diego, CA, USA 3The abbreviations used are: Hp, hairpin; Rz, ribozyme; NTP, nucleoside triphosphate; 8azaA, 8-azaadenosine; 8azaG, 8- azaguanosine; 8azaAMP, 8-azaadenosine 5'-monophosphate; 3'P, 3' cleavage product RNA; 5'R, 5' cleavage product RNA

FIGURE LEGENDS FIGURE 1. A. Structure of the hairpin ribozyme active site with a vanadate mimic of the transition state prepared from the coordinates of Rupert et al. (7) (PDB entry 1M5O). B. General acid base model of the reversible phosphodiester cleavage reaction catalyzed by the hairpin ribozyme. FIGURE 2. Structure and physical properties of 8azaAMP. A. 8azaAMP fluorescence. Fluorescence emission intensity reflects the protonation state of N1 (24). B. 8azaA:U base pair. 8azaA retains the Watson Crick hydrogen bonding face of AMP. FIGURE 3. Enzymatic synthesis of 8azaAMP. (a) Enzymatic synthesis of 8azaATP from ribose and 8-azaadenine: ribokinase (rbsK), 5-phospho-D-ribosyl-α-1-pyrophosphate synthase (prsA), adenine phosphoribosyltransferase (aprT), adenylate kinase (plsA), creatine phosphokinase (ckmT). (b) In situ enzymatic regeneration of catalytic dAMP: adenylate kinase (plsA). (c) In situ enzymatic regeneration of catalytic dADP: creatine phosphokinase (ckmT). (d) Enzymatic dephosphorylation of 8-azaATP to the desired product, 8azaAMP. FIGURE 4. Hairpin ribozyme RNAs. 8azaAHp is a hairpin ribozyme with 8-azaadenosine substituted for adenosine at position 38 that was prepared by ligating 8azaAR (blue) an in vitro transcript with a 5' terminal 8-azaadenosine to ∆A∆BHp48 (green), an in vitro transcript prepared with unmodified NTPs. 8azaHp self-cleaves at the site indicated by the arrow to form a stable complex with a 5’ cleavage product RNA (8azaA5’R) and a 3’ cleavage product (3’P8). P8 and P3A are 3' cleavage product analogs prepared through chemical synthesis that anneal with 8azaA5’R to form H1 helices with 8 or 3 base pairs, respectively. An 8azaAHp variant with 3 base pairs in H1 forms through self-ligation with P3A then cleaves to form an unstable complex that dissociates rapidly during ligation-chase assays. FIGURE 5. A. pH dependence of self-cleavage kinetics. Lines represent fits to Equation 1. B. pH dependence of fluorescence emission intensity. Fluorescence emission intensities were normalized to the maximum emission intensity of unprotonated 8azaA. Lines represent fits to Equation 2 with FBH fixed at 0.23. FIGURE 6. A. Model of the hairpin ribozyme transition state in which A38 acts as a general base catalyst to activate the 5'-oxygen nucleophile and G8 acts as a general acid catalyst to protonate the departing 2'-oxygen during ligation. B. Specific acid base model of hairpin ribozyme catalysis in which A38 and G8 facilitate proton transfer through water.

mailto:[email protected]


12

Figure 1

A-1

A38

G+1G8

NN

N

NN

O

O-O

5'-O A-1

R

NN

NO

N

N

O

OH3'-O

O

PO O-

H

R

G+1

HH

H

NN

N

NN

O

OO

5'-O A-1

R

NN

NO

N

N

O

OH3'-O

O

PO O-

R

G+1

HH H

H

H

NN

N

NN

O

OO

5'-O A-1

R

NN

NO

N

N

O

OH3'-O

O

PO O-

RH

G+1

HH

H

HOH

H

NN

N

NN

O

OO

5'-O A-1

R

NN

NO

N

N

O

OH3'-O

O

PO O

H

RH

G+1

HH

HHA38

G8

H H H

A38 G8 A38 G8A38

G8

A

B

Correspondence of functional and microscopic pKa values in a ribozyme Figure 2

13


14

Figure 3

8-azaA

8-azaAMP

8-azaADP

8-azaATP

Ribose

R5P

PRPP

rbsK

prsA

aprT

plsA

pykF

dATP

dADP

dAMP

PEP

Pyruvate

PPi

dATP

dATP

dADP

(a)

dAMP + dATP 2dADP + 2PEP 2dATP + 2PyruvateplsA pykF

PEP3-PGA 2-PGAgpmA enoA(c)

(b)

(d) 8-azaATP + RiboserbsK / prsA / plsA

8-azaAMP + PRPP


15

Figure 4

CA

P8

GUC

CCU

GU

C 3’

5’

P3A

GUC

CACAAA 3’

5’

Hp

5’R•3’P

G

UG

C

AC

ACA

GU

A

A38G

UU A

GC A

CUGG

GACC

U AA

UGGC

ACCG

U

AU A

AAAGAGUG

A

CA U

G

UGA

AA

GC

A

AAGG

G+1UC

CAC

GA

U

GG

GG

A AACACC

C

A

A

5’

H1

H2H3

H4

H5H6

AG

UA

C3’

AB

G

UG

C

AC

ACA

GU

A

8azaA38G

UU A

GC A

CUGG

GACC

U AA

UGGC

ACCG

U

AU A

AAAGAGUG

A

CA U

G

UGA

AA

GC

A

AAGG

G+1UC

CACCU

GU

C

GA

U

GG

GG

A AACACC

C

A

A

5’ 3’

H1

H2H3

H4

H5H6

AB

8azaAHp

8azaA5’R•3’P8


16

Figure 5

pH

pKa A38 6.0 ± 0.1 8azaA38

8azaAHp 8azaAMP

8azaAHpm

A

B

pKa

4.0 ± 0.2

4.6 ± 0.25.0 ± 0.2

2.5 ± 0.1

k cle

avag

eHp

(min

-1)

kcleavage 8azaAHp (m

in -1)

pH


17

Figure 6

A

NN

N

NN

O

OO

5'-O A-1

R

NN

NO

N

N

O

OH3'-O

O

PO O

H

RH

G+1

HH

HH

NN

N

NN

O

OO

5'-O A-1

R

NN

NO

N

N

O

OH3'-O

O

PO O

H

RH

G+1

HH

HH

O

OH

H

A38G8

A38G8

B

correspondence of functional and microscopic pka values in a ribozyme the ph dependence of

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