Productive folding to the native state by a group II intron ribozyme1

doi:10.1006/jmbi.2001.5233 available online at http://www.idealibrary.com on

J. Mol. Biol. (2002) 315, 297±310

Productive Folding to the Native State by a Group II Intron Ribozyme Jennifer F. Swisher1, Linhui J. Su4, Michael Brenowitz2 Vernon E. Anderson3 and Anna Marie Pyle4,5* 1

Integrated Program in Cellular, Molecular, and Biophysical Studies, Columbia University, New York, NY 10032, USA 2 Department of Biochemistry Albert Einstein College of Medicine, Bronx, NY 10461 USA 3

Department of Biochemistry Case Western Reserve University, Cleveland, OH 44106, USA 4 Department of Biochemistry and Molecular Biophysics and the 5

Howard Hughes Medical Institute, Columbia University New York, NY 10032, USA

Group II introns are large catalytic RNA molecules that fold into compact structures essential for the catalysis of splicing and intron mobility reactions. Despite a growing body of information on the folded state of group II introns at equilibrium, there is currently no information on the folding pathway and little information on the ionic requirements for folding. Folding isotherms were determined by hydroxyl radical footprinting for the 32 individual protections that are distributed throughout a group II intron ribozyme derived from intron ai5g. The isotherms span a similar range of Mg2‡ concentrations and share a similar index of cooperativity. Time-resolved hydroxyl radical footprinting studies show that all regions of the ribozyme fold slowly and with remarkable synchrony into a single catalytically active structure at a rate comparable to those of other ribozymes studied thus far. The rate constants for the formation of tertiary contacts and recovery of catalytic activity are identical within experimental error. Catalytic activity analyses in the presence of urea provide no evidence that the slow folding of the ai5g intron is attributable to the presence of unproductive kinetic traps along the folding pathway. Taken together, the data suggest that the rate-limiting step for folding of group II intron ai5g occurs early along the reaction pathway. We propose that this behavior resembles protein folding that is limited in rate by high contact order, or the need to form key tertiary interactions from partners that are located far apart in the primary or secondary structure. # 2002 Academic Press

*Corresponding author

Keywords: RNA folding; footprinting; kinetics; group II ribozyme; magnesium titration

Introduction Catalytic RNA molecules (ribozymes) are particularly valuable systems for studying RNA tertiary folding because formation of the native state can be sensitively monitored through acquisition of chemical activity. Most of our knowledge about the thermodynamic stability and kinetic folding pathways for RNA tertiary structures comes from two ribozyme systems: the group I self-splicing introns (such as the archetypal Tetrahymena ribozyme1 ± 7) and the catalytic RNA components of bacterial RNase P.8 ± 10 Studies on these and other molecules have shown that the timescale for RNA folding can range from submilliseconds to minutes3,6,10 ± 12 and, with a few exceptions,13 there is usually a requirement for divalent cations (usually E-mail address of the corresponding author: [email protected] 0022-2836/02/030297±14 $35.00/0

Mg2‡ at 1 mM-100 mM). Furthermore, there is often a substantial hierarchy with regard to the stability and folding timescale of substructures within the parent RNA. These substructures represent either obligate (on-pathway) species that occur during folding, or they can represent kinetic traps (off-pathway intermediates) that delay acquisition of the native structure. Slow formation of the native state of RNA has been shown in several cases to be due to the presence of traps, rather than inherently slow events that lie along the folding pathway.2,11,12 Since most of our information about RNA folding comes from a small set of RNA subtypes, it has been of great interest to study the tertiary folding of other large catalytic RNA molecules. Group II introns represent a third structurally distinct class of large ribozymes with unique mechanistic features that have considerably broadened our understanding of RNA enzymology. However, little is # 2002 Academic Press

298 known about the folding mechanism or stability of group II introns and detailed information about their folded structures has only recently begun to develop. A number of group II intron features distinguish them from previously studied ribozymes: group II introns tend to be very large, ranging from 500 to over 1000 nucleotides in length, with an unusually low level of sequence conservation.14,15 Furthermore, the arrangement of tertiary contacts and conserved nucleotides is far more dispersed throughout their secondary structure compared with the group I introns or RNase P.15,16 Finally, group II introns tend to have unusually high ionic requirements for maximal activity.15,17 Given these differences from other ribozymes, it will be interesting to determine if group II introns follow existing models for RNA tertiary folding, or if they are described by a new paradigm. The D135 ribozyme derived from intron ai5g has proven to be a particularly useful system for studying group II intron tertiary structure. This ribo-

Folding of a Group II Intron Ribozyme

zyme contains all the domains demonstrated to contribute to catalysis18 ± 20 and catalyzes multipleturnover hydrolytic cleavage of short oligonucleotide substrates that mimic the 50 -exon. The D135 ribozyme folds into a compact, homogeneous structure with an extensively internalized catalytic core.21 Hydroxyl radical footprinting experiments revealed 32 distinct regions of protection in the D135 molecule in the presence of saturating concentrations of Mg2‡, thereby establishing a compact core structure for the ribozyme and revealing the regions that are most highly internalized within the active molecule (Figure 1). The hydroxyl radical protection patterns are consistent with the speci®c packing arrangements predicted in a recent model of the group II intron core structure, which was built using functional distance constraints from nucleotide analog interference suppression (NAIS) experiments.21 In the present study, we present the ®rst experiments to examine the overall Mg2‡ requirements and folding kinetics of a group II intron ribozyme.

Figure 1. Protection map of D135. A secondary structural representation of the ribozyme showing regions protected from hydroxyl radical cleavage. Color codes represent the degree of protection for each region. Known positions of tertiary interaction are represented by pairs of greek letters. Upper and lower case roman letters (and primes) indicate domains and substructural helices within individual domains, respectively. Reprinted with permission from the EMBO Journal.

299

Using hydroxyl radical footprinting, we have simultaneously visualized the folded state of all nucleotides within the D135 ribozyme as a function of [Mg2‡] and time.3,5,6,22 Titration experiments reveal that ai5g requires high Mg2‡ concentrations for folding and undergoes a highly cooperative folding transition. Kinetic footprinting studies reveal that the catalytic core (and all other substructures) is internalized in a slow and apparently concerted fashion. The formation of the catalytic core occurs coincidently with the acquisition of catalytic activity by D135 on a timescale comparable to that observed for other large catalytic RNAs. We propose that D135 folds directly to the native state and that the slow folding results from a conformational search for a key tertiary interaction early in the reaction pathway.

[MgCl 2 ] mM

U 0 2 4 6 8 10 20 40 60 80 10 0 12 5 15 0 T1

Folding of a Group II Intron Ribozyme

377 363 343 312

256

Results Mg2‡ dependence for the folding of D135 substructures Experimental design The Mg2‡ dependence of D135 folding was monitored by hydroxyl radical footprinting in order to determine if there is a hierarchy in the stability of individual intron substructures and to determine if a speci®c folding event can be linked to the high Mg2‡ requirements that are typical for group II introns (Figure 2). Hydroxyl radical footprinting has provided a sensitive tool for analyzing the kinetics,5,6 and thermodynamics9,10,23 of group I intron and RNase P tertiary folding, and to evaluate the relative contributions of individual domains to the overall tertiary structure.8,24 ± 26 To begin evaluating the stability of individual intronic substructures, D135 was footprinted as a function of MgCl2 concentration in the presence and absence of KCl. MgCl2 titrations were conducted in duplicate and the resultant Y values were quanti®ed as described (see Materials and Methods). The MgCl2 dependence of Y for each protected region was plotted on a semi-log plot and ®t with the twostate binding isotherm (equation (2), Figure 3) in order to obtain KMg and n values for each region of the ribozyme (Table 1). Magnesium dependence of individual D135 footprints One of the most notable features of the data is that no region or subdomain of the intron becomes protected at the low concentrations of MgCl2 that are typical for the folding of other ribozymes and RNA motifs (1-10 mM1,8,27). Instead, the data reveal an overall lack of protection at all intronic positions until  10 mM MgCl2 (Figures 2 and 3), approaching saturation at 60 mM MgCl2. The folding isotherms are all moderately cooperative, with nH values that range from 1.5(0.2) to 2.8(0.4) and cluster around 2.0 (Table 1). The

222 213 207 200

185

171

148 144

130 123

Figure 2. Magnesium titration of the hydroxyl radical footprint of the D135 ribozyme. Shown is a representative gel containing footprinting reactions performed on D135 folded in the presence of indicated concentrations of MgCl2 at 42  C for ten minutes. Gels of varying percentages were used for each set of samples in order to adequately resolve all regions of the D135 molecule. The partial T1 digest of D135 (right) is labeled to indicate the nucleotide numbers corresponding to the products of hydroxyl radical cleavage (the T1 products are shifted up one position relative to radical cleavage products due to the difference in their 30 ends).

small magnitude of the nH values indicates that none of the regions folds in a manner that is highly cooperative with respect to magnesium concen-

300

Folding of a Group II Intron Ribozyme

Figure 3 (legend opposite)

tration. All protected regions of the intron become internalized at approximately the same concentration of MgCl2 (Figure 2) with KMg values ranging from 18(1.3) mM to 47(3.7) mM that cluster around 35.0 (Figure 3; Table 1). Therefore, the overall Mg2‡ dependence is quite similar

among regions, with regard to both the midpoints and steepness of the transitions. The analysis of the Mg2‡ titrations was complicated by the inhibition of hydroxyl radical cleavage of this ribozyme at very high Mg2‡ concentrations. Although this inhibition prevented inclusion of footprinting exper-

Folding of a Group II Intron Ribozyme

301

Figure 3. Representative magnesium-dependence curves for D135 folding. Fractional saturation, Y, of each protected region was plotted as a function of [MgCl2] and ®t to equation (2), which describes two-state binding (Materials and Methods). Representative rate curves are shown for indicated regions. Numerals and Greek letters above plots indicate intronic position and tertiary interactions, respectively.

iments performed above 300 mM MgCl2, suf®cient data were acquired to de®ne the upper limit of the folding transition. Remarkably, the presence of 500 mM KCl has very little effect on the magnesium requirements

for folding. Magnesium titrations performed in the absence of monovalent ion yield similar values for KMg and nH as experiments in which KCl is included (data not shown). This is interesting, since monovalent ions have not been observed to

302

Folding of a Group II Intron Ribozyme

Table 1. Magnesium dependence (KMg), cooperativity (nH), and ®rst-order rate constants (kobs) for the folding of protected regions of ribozyme D135 Region no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32a 32b 32c 32d

Positions

KMg (mM)

nH

kobs (minÿ1)

1-8 13-14 24-26 45-47 56-59 66-77 84-86 96-101 110-116 121-122 129-131 141-143 178-189 198-203 209-213 224-232 312-324 330-342 355-359 371-373 382-386 393-395 399-401 410-415 421-424 584-590 596-601 607-610 620-621 659-665 674-676 819-820 824-825 829-831 837-838

33.8  2.2 35.1  2.7 24.4  1.4 18.4  1.3 22.2  1.0 31.7  2.4 47.0  2.3 47.0  3.7 31.0  1.4 20.0  1.5 31.0  1.7 33.6  1.9 36.6  4.7 33.4  1.8 35.8  2.5 37.6  4.3 35.3  2.1 38.2  2.3 41.8  3.2 31.7  2.1 34.4  1.9 34.7  1.6 35.6  2.1 40.5  1.0 23.3  1.6 44.4  2.0 32.2  2.3 43.3  1.2 23.3  2.5 44.0  2.3 30.8  4.2 44.0  3.4 32.5  2.5 35.2  2.1 39.7  2.7

1.5  0.2 1.8  0.2 1.9  0.2 1.9  0.2 2.2  0.4 1.8  0.3 2.0  0.4 2.0  0.2 2.2  0.3 1.7  0.4 1.8  0.4 1.7  0.3 1.8  0.2 1.9  0.3 1.5  0.2 1.7  0.3 1.7  0.3 1.8  0.5 2.0  0.4 2.1  0.2 1.9  0.2 2.1  0.2 2.1  0.1 1.8  0.3 2.3  0.4 2.1  0.3 1.9  0.3 2.0  0.2 1.7  0.3 2.3  0.2 2.8  0.4 2.2  0.2 2.0  0.2 2.3  0.2 2.2  0.3

1.4  0.2 1.8  0.4 2.3  0.6 1.9  0.4 1.6  0.4 1.8  0.5 2.0  0.3 1.8  0.4 2.2  0.3 3.0  0.3 2.1  0.4 3.0  0.7 2.1  0.5 1.8  0.3 2.3  0.5 2.5  0.4 0.9  0.2 1.2  0.4 2.7  0.7 1.5  0.3 1.3  0.2 0.9  0.3 2.4  0.4 1.2  0.2 1.8  0.3 2.0  1.0 1.9  0.2 1.4  0.5 1.2  0.2 1.5  0.7 2.8  0.9 1.4  0.1 1.2  0.2 1.1  0.1 1.4  0.1

in¯uence the steady-state footprinting pattern of the ribozyme, even though they are known to affect catalytic activity and the homogeneity of folding (28; L.J.S and A.M.P., unpublished results).

capturing the D135 folding pathway under these experimental conditions.

Timescale for the formation of D135 footprints

Peroxonitrite was added to end-labeled D135 at various times after the initiation of folding, and the hydroxyl radical cleavage products were visualized on denaturing polyacrylamide gels (Figure 4). The most notable feature of the resultant timecourse is that all 32 protections in D135 appear with remarkable synchrony (Figure 4). When these data are quanti®ed, the rate constants for internalization of D135 core elements (formation of the protections) fall within a narrow range of values ranging from 0.9(0.2) to 2.8(0.9) minÿ1 (Figure 5, Table 1). The timecourses for protection of all regions were readily ®t by a single-exponential equation, suggesting a uniform kinetic process from a single population (Figure 5, Table 1). A particularly unusual aspect of the data is that all the footprints form on a very slow timescale. At ®ve to ten seconds following the addition of Mg2‡, virtually no hydroxyl radical protection is observed (Figure 4). Appreciable protection is not observed until 30 seconds for all 32 regions. Protection patterns are ®rmly established by

Experimental design When the hydroxyl radical footprints of an RNA are monitored as a function of Mg2‡ concentration, one obtains information about structural states of the ribozyme at equilibrium. By contrast, when an RNA is footprinted as a function of time after initiation of folding, one obtains structural snapshots of the molecule at various stages along its folding pathway. This approach requires the generation of hydroxyl radicals at a timescale that is far more rapid than individual RNA folding events. There are several methods for the rapid generation of hydroxyl radicals: synchrotron radiation for millisecond timescales6 and peroxonitrite22,29,30 and Fe-EDTA31 for slower timescales of the order of tens of seconds to minutes. The decomposition of peroxonitrite to hydroxyl radicals takes place with a half-life of 100 ms under the buffer conditions used for these studies (V.E.A., data not shown), which is more than adequate time resolution for

Kinetic behavior of D135 footprints

303

Time (seconds)

U 0 3 5 10 20 30 40 60 90 120 150 300 600 T1

Folding of a Group II Intron Ribozyme

172 149 131 119 111 97,98 87 85

Given the unusually slow rate constants observed for folding, it was necessary to compare the behavior of D135 with a control RNA that is known to fold rapidly. We therefore studied folding of D135 side-by-side with the L-21 Sca1 ribozyme from Tetrahymena thermophila, which has a well-de®ned folding pathway that includes rapid folding transitions.2,5 In agreement with published data, we observe that the Tetrahymena ribozyme folds rapidly, resulting in a majority of regions that are fully protected by the time hydroxyl radicals are generated by peroxonitrite (t1/2  0.1 second, data not shown22). Therefore, the slow kinetics of D135 folding are readily monitored using peroxonitrite as the footprinting reagent and are not an artifact of the experimental approach.

73

Monitoring folding through D135 ribozyme activity

46 45

14

10

Figure 4. Time-resolved hydroxyl radical footprinting of the D135 ribozyme. Representative gel containing footprinting reactions performed at the indicated times after the addition of 100 mM MgCl2 at 42  C. Samples were run on several gels of different polyacrylamide concentrations for adequate resolution of the entire D135 molecule. A partial T1 digest of D135 (left) is labeled as in Figure 1.

40-60 seconds (Figure 4), resulting in the apparent rate constants described above (Figure 5, Table 1). This ®nding is unusual, in that all RNA molecules studied so far have at least some structural elements that form protected motifs on a timescale that is much faster than one minute.3,6,10

In order to complement the footprinting studies and to relate the rate of D135 folding to the kinetics of ribozyme activity, we examined the rate at which D135 acquires its native structure via acquisition of activity assays. These experiments are conducted by monitoring ribozyme activity immediately after initiating folding with Mg2‡. Experiments of this type serve two purposes: they provide an independent estimate of the folding rate constant and they help to establish whether the tertiary structure that is re¯ected through footprinting represents the active conformation of the ribozyme. Using this approach, previous studies have shown that a group I intron ribozyme folds rapidly to a mixture of active and inactive conformations4,12 under the reaction conditions of the original time-resolved footprinting studies.6 As a result, kinetic traps along the folding pathway signi®cantly delay acquisition of the native state.4,12,32,33 Thus, a ribozyme may acquire catalytic activity more slowly than the formation of hydroxyl radical protections. The assay for acquisition of activity requires a multiple-turnover ribozyme reaction that is conducted under conditions in which folding, rather than chemical catalysis, is rate limiting during the ®rst turnover of reaction. To determine reaction conditions with a suf®ciently rapid chemical rate, a burst kinetics experiment was ®rst conducted with folded ribozyme. Ribozyme was pre-folded in Mg2‡, and reaction was initiated by the addition of substrate oligonucleotide. At slightly elevated pH (8.0), the rate constant for oligonucleotide cleavage by D135 becomes rapid (kobs  3 minÿ1, Figure 6(a)) and faster than the folding rates observed during the footprinting studies (1 minÿ1, Figure 5). Having established conditions under which chemistry is faster than folding (pH 8.0), the assay for acquisition of activity was then carried out. Both MgCl2 and excess substrate were supplied simultaneously to unfolded ribozyme and timepoints were taken at indicated intervals (Figure 6(b)). When multiple-turnover experiments

304

Folding of a Group II Intron Ribozyme

Figure 5 (legend opposite)

are conducted in this way, the resultant burst phase should represent the rate constants of D135 folding,4 rather than catalysis as set up previously with prefolded ribozyme (Figure 6(a)). After isolating reaction products and quanti®cation, the data revealed a burst phase followed by the slow phase that is comparable to the rate constant of product release. In this case, however, the burst rate

constant was 1.01(0.17) minÿ1 rather than the 3.0(0.10) minÿ1 observed when the ribozyme was prefolded in Mg2‡. This value (kfold ˆ 1.01 minÿ1) is in very good agreement with the lower limit of the rate constants for D135 tertiary folding provided by the footprinting assay (kobs ˆ 0.90 (0.20) minÿ1, Table 1). The folding conditions used in the ribozyme activity assay were identical

Folding of a Group II Intron Ribozyme

305

Figure 5. Representative kinetic progress curves for D135 folding. Fractional saturation of each protected region was plotted as a function of time and ®t to a ®rst-order rate equation as described in Materials and Methods. Representative rate curves are shown for indicated regions. Numerals and Greek letters above the plots indicate intronic position and tertiary interactions, respectively.

with those used in footprinting assays, with the exception of a higher pH. The parity between these results indicates that D135 acquires catalytic activity as soon as it folds into the structure re¯ected by the footprinting experiments. This behavior has not been typical for other ribozymes

studied to date. Lastly, the observed burst amplitude of 1.0 (Figure 6(b)) re¯ects homogeneous folding to the active structure with a single rate constant, suggesting that an alternative pathway to the native structure is unlikely under these experimental conditions.

306

Folding of a Group II Intron Ribozyme

Typically, when the folding rate speeds up in the presence of subdenaturing urea concentrations, native folding is inhibited by a form of kinetic trap.4,12 Conversely, if the folding rate is constant or slows in the presence of subdenaturing urea, off-pathway kinetic traps are contraindicated.10,34 The burst rate constant of D135 was examined throughout a range of urea concentrations. At values of 0.05, 0.25, and 0.5 M urea , the burst rate constant was found to vary from 0.85 to 1.2 minÿ1 (Figure 6(c)). The limit for subdenaturing urea is 1 M, as determined by full steady-state urea titrations (L.J.S. and A.M.P., unpublished results), and it is therefore not surprising that above 0.75 M urea, kobs becomes noticeably reduced (data not shown). Together, these data suggest that a kinetic trap does not limit the D135 folding rate.

Discussion

Figure 6. Native folding of the D135 ribozyme. (a) Ribozyme D135 (1 mM) was folded in 100 mM MgCl2 for ten minutes at 42  C (pH 8.0), before initiation of the cleavage reaction with the addition of several equivalents of substrate. Three independent experiments yielded an average burst rate constant of 3.0(0.10) minÿ1, which represents the rate of chemistry (based on comparative rate constants with a modi®ed substrate). The observed rate constant for subsequent turnovers was 0.14(0.07) minÿ1, which is comparable to the rate of product release measured via pulse-chase experiments (L.J.S. and A.M.P., data not shown). Dotted dark gray and light gray lines represent burst rate constant and size of active D135 population, respectively. (b) Ribozyme folding and substrate cleavage reactions were initiated concomitantly with the simultaneous addition of MgCl2 and substrate. Three independent determinations yielded an average burst rate constant of 1.01(0.17) minÿ1. (c) The same as in (b), but in the presence of 0.5 M urea. A burst rate constant of 0.88(0.15) minÿ1 was observed.

To further interpret factors that might limit the folding rate constant, the folding burst was examined in the presence of increasing amounts of urea.

Based on previous work with many different functional RNA molecules and ribozymes, it has been assumed that slow folding of an RNA to the native state is due to the presence of kinetic traps that delay acquisition of the ®nal structure. This model implies that RNA domains collapse rapidly into native folds, or into inactive substructures that must slowly be rearranged in order to reach an active state of the molecule. This is because many functional RNA molecules are assembled from independently folded modules with distinct ionic and temporal requirements for folding and misfolding. D135 is the ®rst example of a large, slow-folding RNA that appears to proceed directly to a fully active state. It is unimpeded by kinetic traps and acquires activity as soon as it folds into a de®ned structure. Based on this behavior, it is useful to think of D135 as an RNA that folds slowly, but accurately. This strategy allows D135 to acquire its native conformation on the same timescale as other large ribozymes, despite folding behavior that differs dramatically from that of group I- or RNase P-derived ribozymes. For example, the L-21 ScaI ribozyme derived from the Tetrahymena group I intron experiences structural collapse >20 times more rapidly than the format of its native structure.35 Individual substructures within the L-21 ribozyme fold even faster.6 Nonetheless, L-21 ScaI reaches its native conformation at nearly the same rate as D135 (1.5 minÿ1 for L-21 versus 1.0 minÿ1 for D135;2,4 and Figure 6(b)). This difference is due to initial misfolding of L-21 ScaI and re¯ects its need for structural rearrangement in order to reach the active conformation. Folding of L-21 to the native state can be accelerated by the addition of subdenaturing concentrations of urea,11 which reduces the activation energy for secondary structural rearrangements and allows the ribozyme to escape kinetic traps in its folding pathway.4,12 The folding of the RNA subunit of RNase P is similar in this

Folding of a Group II Intron Ribozyme

regard:36 it accomplishes tertiary collapse quickly in certain regions, but attains catalytic activity on a much slower timescale. Based on the D135 folding timecourses and the lack of kinetic traps (evident from the fact that folding rate does not accelerate in urea), there are three possible models to explain the D135 folding pathway. (A) Folding of D135 could occur by a slow, two-state mechanism from unfolded to the native state. (B) There may be ¯eeting intermediates along the pathway that could not be detected or were too diverse in conformation to be examined by these methods. (C) There may be an onpathway intermediate during D135 folding. In the latter case, the transition from the unfolded state (U) to the intermediate (I) would necessarily be slow, while the transition from I to the native state (N) would be rapid. An example of the latter behavior is provided by the folding of C domain from RNase P RNA.10 The rapid acquisition of activity by this RNA and the deceleration of its folding in the presence of urea have been used as evidence that kinetic traps do not represent a compulsory feature of RNA folding. And like D135, the timecourse for folding at saturating Mg2‡ is uniform.10 What distinguishes D135 from C domain is that the latter folds much more rapidly (6.5 sÿ1), and it is signi®cantly smaller than D135 (C domain: 225 nucleotides, D135: 618 nucleotides). In fact, the folding rate constant of C domain is not all that different from the folding rate of other stable RNA substructures that have been studied previously (60 minÿ1 for P456, 120 minÿ1 for P5abc).3,6 The folding mechanism of group I intron bI5 is similar to that of C domain, although bI5 collapses rapidly to an on-pathway intermediate that rearranges slowly into the native conformer.34 Thus, whether D135 folds directly to the native state or through an on-pathway intermediate, comparisons with C domain and bI5 highlight the major question raised by the present study: why does an RNA as large and complex as D135 fold directly to a productive state on such a slow timescale? Answers to this question may lie with recent analyses of protein folding rate constants.37 By comparing the folding kinetics with the topological complexity of folded proteins, Baker and colleagues have shown that there is a good correlation between intrinsic folding rate constant of a protein and its ``contact order'', which is a measure of the relative contribution of local (proximal) and nonlocal (distant) interaction partners in the protein sequence.38 This correlation indicates that molecules containing tertiary interaction partners that are close together in primary sequence will fold fast. Conversely, molecules undergo slower folding if they are composed of interaction partners that are located far apart in primary sequence. It is interesting to consider the parallel between group II intron tertiary structure and a protein with many long-range interactions (high ``contact order''). Many investigators have pointed out that

307 the secondary structure of group II introns is unusually extended, particularly when compared to other ribozymes.16,39 Furthermore, the conserved nucleotides and important functional groups in group II introns occur in scattered clusters located far apart in the secondary structure.15 Perhaps most important, the tertiary interactions that have been characterized to date contain pairs of interacting residues that are separated by very large distances in primary and secondary structure.40 Thus, upon close inspection, group II introns appear to be biopolymers with a high contact order. Like analogous proteins, group II introns may therefore fold slowly because of the large entropic barrier to the formation of its far-¯ung tertiary contacts. In addition to aiding interpretation of the kinetic studies, the Mg2‡ titrations provide important information about the tertiary structure of D135. It has long been known that almost all group II introns require high concentrations of Mg2‡ for optimal activity, but this requirement might be traced to a role in exon binding, catalysis, or internal conformational rearrangement. This work establishes that high Mg2‡ is speci®cally required for folding of the intron, and not necessarily any downstream function. But perhaps the most notable feature of the Mg2‡ titration data is its striking uniformity: all regions fold at the same concentration of Mg2‡, and no intronic substructures or domains were internalized at lower concentrations of Mg2‡ that fell below that global KMg of 40 mM. This implies that there are no stable tertiary substructures in D135 that can internalize and collapse at the [Mg2‡] commonly seen for protection of other small RNA motifs. For example, there is no substructure similar to P5abc (a small, stable group I intron motif) in D135; regions like D3 or small subsections of D1 do not adopt a collapsed form on their own. Rather, they appear to be designed to take their place within the intronic architecture only after a pivotal structural event causes overall structural condensation. When considering both the kinetic and Mg2‡ dependence data together, the most plausible model for D135 folding involves the formation of a single ``linchpin'' interaction. This contact is likely to involve at least two elements that are widely separated in the secondary structure (thus explaining the slow folding rate), and the resultant interaction is not very stable (thus explaining the high [Mg2‡] requirement). All other long-range interactions within D135 can form only after the linchpin element has formed, thereby generating a conformation that nucleates folding of the entire intron (a rate-limiting, on-pathway intermediate). Importantly, when the [Mg2‡] has become high enough to stabilize the linchpin contact, it is already so high that subsequent contacts do not require any additional magnesium, and they rapidly condense. As a result, folding appears to be concerted, although it is likely to result from sequential events.

308 This model has several important implications for future research. First of all, the group II intron folding model proposed herein will probably be general if it is, in fact, correct. This is because the basic group II intron secondary structure and the wide dispersal of tertiary interaction partners are universal features of group II introns. Thus, it will be important to test the folding kinetics and Mg2‡ dependencies of other group II introns, with particular attention to the small class that folds at low [Mg2‡] (such as a group II intron from Pylaiella littoralis).39 It will also be interesting to study the folding of constructs that contain other domains. While D135 contains all the motifs that are critical for catalysis, other regions, particularly the 50 -exon, may have important roles in the folding pathway. It will be vital to determine the identity of the putative linchpin interaction in D135 derived from the ai5g intron. Manipulation of this interaction may alter the folding rate constants and Mg2‡ dependence of intron RNA. The ultimate test of whether high contact order limits the overall rate constant for group II intron folding (and indeed whether this paradigm can be extended to the folding of RNA tertiary structure from secondary structure) will be to vary the distance between partners in the linchpin interaction, once it has been identi®ed. Finally, it will be interesting to determine if there is a role for intron-encoded proteins and other known protein cofactors of group II introns in reducing the translational entropy between pairing partners in the putative linchpin interaction. Through these experiments, group II introns may ultimately reveal new paradigms both for RNA folding and for the role of cognate chaperone proteins.

Materials and Methods RNA preparation The ribozyme variant used in this study (henceforth called D135) spans domains 1-5 of yeast mitochondrial intron ai5g. The ribozyme was transcribed from plasmid pQL71 (also referred to previously as pT7D13521); it begins at the ®rst nucleotide of the intron and incorporates hairpin loops in place of domains 2 and 4.21 In this construct, the terminal linker sequence downstream from D5 is followed by a polylinker which, when cleaved with HindIII and transcribed, results in an extended 35 nucleotide tail that facilitates mapping the 30 end of the ribozyme. Oligonucleotide substrate, 50 -CGUGGUGGGACAUUUUC^GAGCGGU-30 , was synthesized according to standard methods.41 RNA stock solutions were prepared in a buffer of 40 mM Mops (pH 6.0), 10 mM EDTA. RNA was stored at ÿ80  C in 10 mM Mops (pH 6.0), and 1 mM EDTA in order to reduce nonspeci®c cleavage. Hydroxyl radical footprinting reactions D135 was labeled either at the 50 end with [g-32P]ATP using T4 polynucleotide kinase (NEB) or the 30 end42 using a complementary DNA oligonucleotide and DNA polymerase (NEB). Footprinting reactions were carried

Folding of a Group II Intron Ribozyme out at 42  C with 2-4 nM end-labeled D135 in 80 mM Mops (pH 6.0) with speci®ed MgCl2 concentrations in a ®nal reaction volume of 20 ml. In most cases, 500 mM KCl was also contained in the reaction buffer, as indicated. RNA stock solutions (16 ml each) in 80 mM Mops, with or without 500 mM KCl (®nal concentration), were heated to 95  C for one minute. For magnesium titration experiments, 4 ml of a 5  MgCl2 stock was added to each sample to initiate the folding reaction, which was allowed to proceed at 42  C for ten minutes before addition of peroxonitrite footprinting reagent. The concentration of MgCl2 stock solutions ranged from 0.5 mM to 3.5 M in these experiments (resulting in ®nal sample concentrations of 0.1700 mM MgCl2). Time-resolved footprinting experiments were initiated by adding 4 ml of 0.5 M MgCl2 to D135, resulting in a ®nal concentration of 100 mM MgCl2. The unfolded control was mixed with 4 ml of water. Upon addition of MgCl2, each sample was allowed to fold at 42  C for a speci®ed time (from three to 600 seconds) prior to mixing with 8.4 ml of potassium peroxonitrite stock (100 mM in 0.1 M KOH, allowed to pre-heat at 42  C), which resulted in a ®nal peroxonitrite concentration of 30 mM. After mixing, the peroxonitrite footprinting reaction was allowed to proceed for ®ve seconds. The samples were then placed on ice and precipitated by adding sodium acetate to 30 mM, followed by three volumes of ethanol. Products of the footprinting reactions were resolved on polyacrylamide or long ranger (FMC Bioproducts) gels of varying concentrations (polyacrylamide, 5-20 %, long ranger, 5-7 %), cast on custom-made 3600 plates. Dried gels were analyzed and products quanti®ed using a Molecular Dynamics Storm 840 PhosphorImager. Quantitative analysis of footprinting data The intensity of end-labeled cleavage products was quanti®ed for each protected region using ImageQuant software IQ v1.2 (MacAffe). As in previous studies,6,21 a given protected region (rather than an individual band) was speci®ed, or boxed, and the cpm for that region was measured in all lanes. The same region was also chosen from a control lane that was designated to provide ``background'' counts. This background lane contained a fully folded sample (100 mM MgCl2 for ten minutes at 42  C) that was not treated with peroxonitrite; the cpm in any given region of this lane represents the background RNA degradation in the sample prior to addition of the footprinting reagent. Values for cpm in the background sample were subtracted from values for corresponding regions in the experimental lanes. These corrected intensities were then normalized using a reference region from the sample lane in order to account for small differences in loaded counts. Reference regions represent stretches of nucleotides that do not display a trend in intensity with increasing time or magnesium concentration. Each protected region was normalized with a relatively proximal reference (within 100 nucleotides). These normalized intensity values (X) were then used  at each region, to calculate the fractional saturation (Y) according to equation (1): Y ˆ …X-LL†=…UL-LL†

…1†

The fractional saturation value Y describes the protection at a region relative to the protection observed in the

309

Folding of a Group II Intron Ribozyme fully folded sample (LL, or lower limit of cleavage intensity) and compared to the unfolded sample (UL, or upper limit of cleavage intensity), all of which were treated with peroxonitrite. To analyze the Mg2‡ titration data, Y values (fraction protected, Figure 2) for a given region were plotted against the logarithm of [MgCl2]. Each semi-log plot was composed of data sets from two independent experiments. The combined data were ®t to a cooperative saturation curve:43 Y ˆ

‰MgŠn ‡ ‰MgŠn

n KMg

…2†

where the KMg term is related to an af®nity constant but speci®cally refers to the magnesium concentration at which the region is half-protected (Y ˆ 0.5) and the coef®cient n provides both a relative measure of cooperativity and an estimated lower limit for the number of magnesium ions involved. Kinetic footprinting analyses were carried out similarly by plotting values for the fraction protected versus time and ®tting the data to the ®rst order rate equation: Y ˆ 1 ÿ eÿkt

…3†

where Y is the fractional saturation of a given site at time, t, and k is the ®rst-order rate constant (minÿ1). Assay for the acquisition of catalytic activity The oligonucleotide substrate for all ribozyme activity assays was 50 end-labeled with [g-32P]ATP and its concentration was determined from speci®c activity. Concentrations of unlabeled substrate and ribozyme molecules were determined using a Hewlett-Packard HP 845X UV-visible spectrophotometer. For the multipleturnover ribozyme reactions performed in this study, only a small fraction of the substrate molecules were radiolabeled: in general, 1 ml of 100 nM 32P-labeled substrate was combined with 99 ml of unlabeled substrate (100 M) to create a 10 stock of concentrated, tracelabeled substrate. All ribozyme cleavage experiments were performed at 42  C in a total reaction volume of 70 ml , which contained trace D135 and excess oligonucleotide substrate, in a buffer of 80 mM Hepes (pH 8.0), 500 mM KCl, and 100 mM MgCl2. To begin a given experiment, separate RNA stock solutions containing ribozyme and substrate (35 ml each in 80 mM Hepes and 500 mM KCl), were heated to 95  C for one minute and then transferred to a waterbath set at 42  C. For multiple-turnover burst experiments on fully folded populations of D135, both ribozyme (1 mM ®nal) and substrate (4 mM ®nal) were separately equilibrated in 100 mM MgCl2 at 42  C for ten minutes before being combined to initiate reaction. For experiments designed to monitor the rate of folding (acquisition of activity), timepoints were taken immediately after mixing unfolded ribozyme (1 mM ®nal) with substrate (10 mM ®nal), Mg2‡ (®nal concentration 100 mM) and, where applicable, urea (®nal concentration 0.05, 0.25, 0.5, 0.75 or 1 M). At 20 second intervals, aliquots of 2 ml were withdrawn from the cleavage reaction and added to an equal volume of quench buffer. These were analyzed by PAGE and quanti®ed as described previously.20 Similar to related analyses on the Tetrahymena intron,4 product formation was plotted as a function of time, and rate constants for the burst and plateau phases of reaction were determined by ®tting the data as

described previously:21 frac‰PŠ ˆ A1 …1 ÿ eÿk1 t † ‡ A2 …1 ÿ eÿk2 t †

…4†

where P is product, A1 is the amplitude of ®rst turnover (burst), k1 is the rate constant for the ®rst turnover (burst), A2 is the amplitude of subsequent turnovers, k2 is the observed rate constant for subsequent turnovers.

Acknowledgements We thank Qiaolian Liu for constructing the plasmid pQL71 and Rick Russell for helpful discussions. We acknowledge generous support from the NIH to J.S. (Hormones: Biochemistry and Molecular Biology DK 07328), L.J.S. (Biophysics Training Grant T32 GM08281), M.B. (Albert Einstein Center for Synchrotron Biosciences P41-RR01633), and A.M.P. (RO1 GM50313). A.M.P. is an assistant investigator of the Howard Hughes Medical Institute.

References 1. Celander, D. W. & Cech, T. R. (1991). Visualizing the higher order folding of a catalytic RNA molecule. Science, 251, 401-407. 2. Zarrinkar, P. P. & Williamson, J. R. (1994). Kinetic intermediates in RNA folding. Science, 265, 918-924. 3. Deras, M. L., Brenowitz, M., Ralston, C. Y., Chance, M. R. & Woodson, S. A. (2000). Folding mechanism of the Tetrahymena ribozyme P4-P6 domain. Biochemistry, 39, 10975-10985. 4. Russell, R. & Herschlag, D. (1999). New pathways for folding of the Tetrahymena group I RNA enzyme. J. Mol. Biol. 291, 1155-1167. 5. Sclavi, B., Woodson, S., Sullivan, M., Chance, M. & Brenowitz, M. (1997). Time-resolved synchrotron X-ray ``footprinting'', a new approach to the study of nucleic acid structure and function: application to protein-DNA interactions and RNA folding. J. Mol. Biol. 266, 144-159. 6. Sclavi, B., Sullivan, M., Chance, M., Brenowitz, M. & Woodson, S. (1998). RNA folding at millisecond intervals by synchrotron hydroxyl radical footprinting. Science, 279, 1940-1943. 7. Silverman, S. K. & Cech, T. R. (2001). An early transition state for folding of the P4-P6 RNA domain. RNA, 7, 161-166. 8. Pan, T. (1995). Higher-order folding and domain analysis of the ribozyme from Bacillus subtilis ribonuclease P. Biochemistry, 34, 902-909. 9. Fang, X., Pan, T. & Sosnick, T. R. (1999). A thermodynamic framework and cooperativity in the tertiary folding of a Mg2‡-dependent ribozyme. Biochemistry, 38, 16840-16846. 10. Fang, X., Pan, T. & Sosnick, T. (1999). Mg2‡-dependent folding of a large ribozyme without kinetic traps. Nature Struct. Biol. 6, 1091-1095. 11. Rook, M., Treiber, D. & Williamson, J. (1998). Fast folding mutants of the Tetrahymena group I ribozyme reveal a rugged folding energy landscape. J. Mol. Biol. 281, 609-620. 12. Rook, M., Treiber, D. & Williamson, J. (1999). An optimal Mg(2‡) concentration for kinetic folding of the Tetrahymena ribozyme. Proc. Natl Acad. Sci. USA, 96, 12471-12476.

310

Folding of a Group II Intron Ribozyme

13. Shiman, R. & Draper, D. (2000). Stabilization of RNA tertiary structure by monovalent cations. J. Mol. Biol. 302, 79-91. 14. Michel, F., Umesono, K. & Ozeki, H. (1989). Comparative and functional anatomy of group II catalytic introns-a review. Gene, 82, 5-30. 15. Michel, F. & Ferat, J.-L. (1995). Structure and activities of group II introns. Annu. Rev. Biochem. 64, 435-461. 16. Cech, T. R. (1988). Conserved sequences and structure of group I introns: building an active site for RNA catalysis- a review. Gene, 73, 259-271. 17. Jarrell, K. A., Peebles, C. L., Dietrich, R. C., Romiti, S. L. & Perlman, P. S. (1988). Group II intron selfsplicing: alternative reaction conditions yield novel products. J. Biol. Chem. 263, 3432-3439. 18. Grif®n, E. A., Qin, Z.-F., Michels, W. A. & Pyle, A. M. (1995). Group II intron ribozymes that cleave DNA and RNA linkages with similar ef®ciency, and lack contacts with substrate 20 -hydroxyl groups. Chem. Biol. 2, 761-770. 19. Michels, W. J. & Pyle, A. M. (1995). Conversion of a group II intron into a new multiple-turnover ribozyme that selectively cleaves oligonucleotides: elucidation of reaction mechanism and structure/ function relationships. Biochemistry, 34, 2965-2977. 20. Xiang, Q., Qin, P. Z., Michels, W. J., Freeland, K. & Pyle, A. M. (1998). The sequence-speci®city of a group II intron ribozyme: multiple mechanisms for promoting unusually high discrimination against mismatched targets. Biochemistry, 37, 3839-3849. 21. Swisher, J., Duarte, C., Su, L. & Pyle, A. (2001). Visualizing the solvent-inaccessible core of a group II intron ribozyme. EMBO J. 20, 2051-2061. 22. Chaulk, S. & MacMillan, A. (2000). Characterization of the Tetrahymena ribozyme folding pathway using the kinetic footprinting reagent peroxonitrous acid. Biochemistry, 39, 2-8. 23. Ralston, C. Y., He, Q., Brenowitz, M. & Chance, M. R. (2000). Stability and cooperativity of individual tertiary contacts in RNA revealed through chemical denaturation. Nature Struct. Biol. 7, 371-374. 24. Murphy, F. L. & Cech, T. R. (1993). An independently folding domain of RNA tertiary structure within the Tetrahymena ribozyme. Biochemistry, 32, 5291-5300. 25. Loria, A. & Pan, T. (1996). Domain structure of the ribozyme from eubacterial ribonuclease P. RNA, 2, 551-563. 26. Doherty, E. A. & Doudna, J. A. (1997). The P4-P6 domain directs higher order folding of the Tetrahymena ribozyme core. Biochemistry, 36, 3159-3169. 27. Laing, L. G., Gluick, T. C. & Draper, D. E. (1994). Stabilization of RNA structure by Mg ions. J. Mol. Biol. 237, 577-587. 28. Franzen, J. S., Zhang, M., Chay, T. R. & Peebles, C. L. (1994). Thermal activation of a group II intron

29.

30.

31. 32.

33.

34. 35.

36. 37.

38.

39.

40.

41.

42. 43.

ribozyme reveals multiple conformational states. Biochemistry, 33, 11315-11326. King, P., Anderson, V., Edwards, J., Gustafson, G., Plumb, R. & Suggs, J. (1992). A stable solid that generates hydroxyl radical upon dissolution in aqueous solutions: reaction with proteins and nucleic acid. J. Am. Chem. Soc. 114, 5430-5432. King, P., Jamison, E., Strahs, D., Anderson, V. & Brenowitz, M. (1993). ``Footprinting'' proteins on DNA with peroxonitrous acid. Nucl. Acids Res. 21, 2473-2478. Hampel, K. & Burke, J. (2001). Time-resolved hydroxyl-radical footprinting of RNA using Fe(II)EDTA. Methods Enzymol. 23, 233-239. Treiber, D. & Williamson, J. (2001). Concerted kinetic folding of a multidomain ribozyme with a disrupted loop-receptor interaction. J. Mol. Biol. 305, 11-21. Russell, R. & Herschlag, D. (2001). Probing the folding landscape of the Tetrahymena ribozyme: commitment to form the native conformation is late in the folding pathway. J. Mol. Biol. 308, 839-851. Buchmueller, K. L., Webb, A. E., Richardson, D. A. & Weeks, K. M. (2000). A collapsed non-native RNA folding state. Nature Struct. Biol. 7, 362-366. Russell, R., Millett, I. S., Doniach, S. & Herschlag, D. (2000). Small angle X-ray scattering reveals a compact intermediate in RNA folding. Nature Struct. Biol. 7, 367-370. Zarrinkar, P., Wang, J. & Williamson, J. (1996). Slow folding kinetics of RNase P RNA. RNA, 2, 564-573. Plaxco, K., Simons, K., Ruczinski, I. & Baker, D. (2000). Topology, stability, sequence, and length: de®ning the determinants of two-state protein folding kinetics. Biochemistry, 39, 11177-11183. Plaxco, K. W., Simons, K. T. & Baker, D. (1998). Contact order, transition state placement and the refolding rates of single domain proteins. J. Mol. Biol. 277, 985-994. Costa, M., Fontaine, J. M., Goer, S. L. & Michel, F. (1997). A group II self-splicing intron from the Brown Alga Pylaiella littoralis is active at unusually low magnesium concentrations and forms populations of molecules with a uniform conformation. J. Mol. Biol. 274, 353-364. Qin, P. Z. & Pyle, A. M. (1998). The architectural organization and mechanistic function of group II intron structural elements. Curr. Opin. Struct. Biol. 8, 301-308. Wincott, F., DiRenzo, A., Shaffer, C., Grimm, S., Tracz, D., Workman, C. et al. (1995). Synthesis, deprotection, analysis and puri®cation of RNA and ribozymes. Nucl. Acids Res. 23, 2677-2684. Huang, Z. & Szostak, J. W. (1996). A simple method for 30 -labeling of RNA. Nucl. Acids Res. 24, 43604361. Metzler, D. (1977) Biochemistry, the Chemical Reactions of Living Cells, Academic Press, New York.

Edited by D. Draper (Received 29 August 2001; received in revised form 7 November 2001; accepted 8 November 2001)