The maturase encoded by a group I intron from Aspergillus nidulans stabilizes RNA tertiary structure and promotes rapid splicing1

Article No. jmbi.1999.3070 available online at http://www.idealibrary.com on

J. Mol. Biol. (1999) 292, 987±1001

The Maturase Encoded by a Group I Intron from Aspergillus nidulans Stabilizes RNA Tertiary Structure and Promotes Rapid Splicing Yugong Ho and Richard B. Waring* Department of Biology, Temple University, Philadelphia, PA 19122, USA

The AnCOB group I intron from Aspergillus nidulans self-splices, providing the Mg2‡ concentration is 515 mM. The splicing reaction is greatly stimulated by a maturase protein encoded within the intron itself. An initial structural and biochemical analysis of the splicing reaction has now been performed. The maturase bound rapidly to the precursor RNA (kon ' 3  109 Mÿ1 minÿ1) and remained tightly bound (koff 4 0.04 minÿ1). The catalytic step of 50 splice-site cleavage occurred at a rate of up to 11 minÿ1 under single turnover conditions. The maturaseassisted reaction of heat-denatured RNA proceeded at a rate of about 1 minÿ1, arguing that there are early steps of folding that cannot be readily facilitated by the protein. pH analysis revealed a biphasic pro®le with a pKa of 7.0. The rate of the maturase-assisted reaction was independent of the Mg2‡ concentration down to 3 mM. Self-splicing in optimal Mg2‡ (5150 mM) was tenfold slower, in part because of the existence of an equilibrium between folded and partially folded RNA. In contrast, the maturase very effectively stabilized tertiary structure in 5 mM Mg2‡, a noticeable example being an interaction between the P8 helix and a GNRA sequence that constitutes the L2 terminal loop of the P2 helix. Formation of the 50 splice-site recognition helix was assisted by either the maturase or high concentrations of Mg2‡. The maturase was required during splicing so it is not a true chaperone. However, RNase protection assays and kinetic studies suggest that the maturase recognizes and facilitates folding of an intron with limited tertiary structure and even incomplete secondary structure. # 1999 Academic Press

*Corresponding author

Keywords: homing endonuclease; mitochondria; ribozyme; RNA folding; RNA splicing protein

Introduction Group I introns are a class of catalytic RNA molecule. Some can splice in vitro in the absence of any protein factor; this is termed self-splicing (Cech, 1990). In order to be excised from the RNA precursor (pre-RNA), group I introns must fold into a catalytically active tertiary structure. In many cases, a non-physiological, high concentration of magnesium ions is required for optimal self-splicing activity (Garriga & Lambowitz, 1984; Gampel et al., 1989; Jaeger et al., 1991). Even the pro®cient Abbreviations used: pre-RNA, precursor RNA; ORF, open reading frame; 50 SS, 50 splice-site; 30 SS, 30 splicesite; IGS, internal guide sequence. E-mail address of the corresponding author: [email protected] 0022-2836/99/400987±15 $30.00/0

Tetrahymena ribozyme self-splices in vitro under physiological conditions 20 times more slowly than the estimated rate in vivo (Brehm & Cech, 1983). Genetic and biochemical studies indicate that many group I introns require protein factors for ef®cient splicing in vivo (Lambowitz & Perlman 1990). In this work we start to characterize the role in splicing played by a group I intron-encoded protein called a maturase. Many group I introns have an open reading frame (ORF). These frequently encode a DNA endonuclease which can initiate insertion (termed homing; Dujon, 1989) into an allelic site that lacks the intron. Homing endonucleases fall into four classes, one of which contains one or two copies of a LAGLIDADG amino acid motif (Belfort & Roberts, 1997). Mutational analysis in vivo has shown that certain intron-encoded LAGLIDADG # 1999 Academic Press

988 proteins play a role in splicing their cognate introns and occasionally other introns (reviewed by Lambowitz & Perlman, 1990; Lambowitz et al., 1999). This kind of protein is termed a maturase (Lazowska et al., 1980). However, despite being identi®ed genetically prior to the discovery of selfsplicing introns and catalytic RNA, the functions of maturases, their phylogenetic distribution and their relationship to homing endonucleases are only poorly understood at present. Progress on all three of these issues has been hindered until recently by the lack of a biochemical assay for maturase function. The group I intron in the mitochondrial apocytochrome b gene (AnCOB) in Aspergillus nidulans self-splices in vitro at a Mg2‡ concentration of 15 mM or higher (Hur et al., 1997). AnCOB contains an ORF which is in-phase with the upstream 50 exon. We have reported previously that the intron encodes a maturase which speci®cally and ef®ciently activates AnCOB intron splicing at 5 mM Mg2‡ in vitro (Ho et al., 1997). These experiments established for the ®rst time that a maturase plays a direct role in splicing. Interestingly, the AnCOB protein is also a DNA endonuclease (Ho et al., 1997). The relationship between maturases and homing DNA endonucleases has been discussed in several reviews (Lambowitz & Perlman, 1990; Belfort & Roberts, 1997; Lambowitz et al., 1999). All known group I maturases have two LAGLIDADG motifs. The phylogenetic distribution of LAGLIDADG endonucleases is widespread, but that of the maturases is unknown. Most maturases have been identi®ed in various strains and species of yeast through the isolation of mutants defective in splicing mitrochondrial gene transcripts. Since this genetic approach is not feasible in many organisms, the establishment of a biochemical assay is an important development for addressing this issue. Group II introns also encode proteins which facilitate RNA splicing (Lambowitz & Perlman, 1990), but they are not detectably similar to those from group I introns. An in vitro splicing assay has recently been developed for a group II maturase (Matsuura et al., 1997). The folding pathway of group I introns includes several intermediates and involves the sequential formation and stabilization of ten or more paired helical regions P1-P10 (for labelling conventions see the legend to Figure 9); some secondary structure may fold after or concurrent with tertiary interactions. The P4-P6 region of the Tetrahymena intron folds as an independent unit and is the most thermodynamically stable domain in the intron (Latham & Cech, 1989; Celander & Cech, 1991; Murphy & Cech, 1993). It is also the ®rst higherorder structure to form on the kinetic pathway of RNA folding, being established considerably more rapidly than the P7-P3-P8 domain (Zarrinkar & Williamson, 1994; Downs & Cech, 1996; Sclavi et al., 1998; Treiber et al., 1998).

Maturase-assisted Splicing of a Group I Intron

Maturase sequences that are known to affect RNA splicing have been identi®ed in three (IB1, IB4 and ID) of the 11 group I subgroups de®ned by Michel & Westhof (1990). The AnCOB intron has a P4-P6 domain and belongs to subgroup IB4. Three structures contribute to the stability of the P4-P6 domain of the Tetrahymena intron (subgroup IC1): the P5abc three-way junction, the A-rich bulge (between P5ai and P5aii shown in Figure 9) and a GNRA-tetra loop receptor interaction between the loop of P5b and the P6 extension (Murphy & Cech, 1993, 1994; Costa & Michel, 1995; Cate et al., 1996). The AnCOB intron only retains the A-rich bulge, suggesting that it may be inherently less stable than the Tetrahymena intron and hence more dependent on the assistance of a protein. Group I intron splicing factors fall into three classes: RNA chaperones, nuclear-encoded splicing factors and intron-encoded proteins. All the identi®ed protein factors facilitate folding of the RNA into a catalytically active higher-order structure (reviewed by Weeks, 1997). The Escherichia coli ribosomal S12 protein is an RNA chaperone which promotes RNA folding but is not required during the splicing reaction (Coetzee et al., 1994). A tyrosyl synthetase (CYT-18) in Neurospora crassa binds to intron secondary structure, initially in the P4-P6 region, and then nucleates and facilitates folding of the tertiary structure (Saldanha et al., 1996; Caprara et al., 1996). The yeast Cbp2 protein binds to partially folded tertiary structure, trapping the transiently associated P5-P4-P6 and P7-P3-P8 domains. Cbp2 also facilitates docking of the 50 domain of the intron (P1 and P2) to the conserved core structure (Weeks & Cech, 1995, 1996; Shaw & Lewin, 1995). We therefore wished to determine if the maturase behaved like an RNA chaperone, like CYT-18 or Cbp2, or whether a novel mechanism needed to be proposed. Our initial data suggest that the maturase acts like CYT-18. In this work we perform a preliminary characterization of the rapid and tight binding of the AnCOB maturase to its cognate intron and its striking impact on the splicing reaction. Our data suggest that the maturase recognizes and rapidly folds an intron RNA with limited tertiary structure and even incomplete secondary structure in 25 mM Mg2‡.

Results kobs under single turnover conditions reflects the rate of the forward catalytic step of 50 splice-site cleavage The standard maturase-assisted reaction was performed in 5 mM MgCl2 under single turnover conditions with protein in excess and prebound to the RNA (see Materials and Methods; Figure 1(a)). Before studying the reaction further, we wished to know which step(s) in the splicing pathway contributed to the observed reaction rate. Under

989

Maturase-assisted Splicing of a Group I Intron

stoichiometric burst (1:1) of RNA splicing followed by very slow turnover of the maturase with a rate constant of about 0.006 minÿ1 (Ho et al., 1997). Throughout this work the reaction analyzed will be the ®rst step of splicing. The overall splicing reaction can be described by: kon

‡G

k2

k3

kÿ2

kÿ3

5E-I-3E ‡ M „ 5E-I
M „ 5E
G-I-3E
M „ koff

k4

5E-3E
G-I
M ! 5E-3E ‡ G-I ‡ M Scheme 1

Figure 1. The maturase-assisted splicing reaction in 5 mM Mg2‡. (a) A standard reaction including prebinding of the RNA-maturase complex was performed. PRE is precursor, I3E is intron attached to 30 exon. LE is ligated exons, I is excised intron. The 50 exon is short and not shown; its presence exactly mirrors that of the I3E intermediate, being generated during 50 SS cleavage and disappearing as the RNA molecules complete the second step. (b). The data from (a) were ®tted to a double exponential equation (see Materials and Methods) and yielded the equation Fpre ˆ 0.82eÿ6.4t ‡ 0.13eÿ0.08t ‡ 0.05. The slow phase (undergone by 0.13 of the pre-RNA) and the unreacted material (0.05) were subtracted and just the fast phase was plotted; about 15 % of the slow phase (3 % total pre-RNA) reacted during the ®rst three minutes. The rate constants for the fast and slow phases were 6.4 and 0.08 minÿ1. Squares, pre-RNA; triangles, I3E; diamonds, average of LE and I.

standard conditions, typically, about 80-85 % of the precursor RNA (pre-RNA) reacted very rapidly, 10-15 % reacted 30-100 times slower and 5 % did not react. This was observed with different preparations of both protein and RNA. The maximal rate with saturating protein varied from 5 to 11 minÿ1 over several lots of protein. No multiple turnover experiments are reported here. We have previously shown that there is a

Group I introns react by two sequential transesteri®cation reactions: ®rst, guanosine or GTP attacks the 50 splice-site (50 SS) and is transferred to the ®rst nucleotide of the intron. The exposed 30 -OH end of the upstream exon then attacks the 30 splice site (30 SS), to generate the intron and ligated exons (Cech, 1990). 5E
G-I-3E represents the intermediate produced after cleavage of the 50 SS by G, where G-I-3E (usually abbreviated to I3E) is the G-tagged intron attached to the 30 exon. M signi®es maturase. It is assumed that G cannot readily bind until the RNA is stabilized by the maturase (see below). The binding step (re¯ected by kon and koff) is not involved in the standard reaction as the RNA-protein complex is already formed and binding is very tight (see below). As de®ned, k2 could theoretically represent a slow folding step subsequent to binding and prior to formation of the bimolecular complex in its equilibrated state. It will be shown later that this is not the case, and that k2 represents the rate constant for a rate-limiting step in 50 SS cleavage which occurs after formation of the bimolecular complex. Since the concentration of GTP is saturating, the reaction of the bimolecular complex is pseudo®rst-order. The 50 SS cleavage of the pre-RNA (Figure 1(a)) was ®tted to a biphasic exponential equation (see Materials and Methods). Figure 1(b) shows the reaction pro®le of the faster phase after subtraction of the slower second phase and the unreacted portion. The data argue that k2 > kÿ2 since at t ˆ 0.33 minute, the intermediate, I3E, is still showing a net accumulation despite the fact that the ratio of I3E to precursor is about 6:1 and I3E is also being depleted at a signi®cant rate by undergoing the second splicing step. The rapid depletion of I3E argues that the rate of exon ligation is faster than the reverse of 50 SS cleavage (k3 > kÿ2). Since the level of I3E dropped to a low level as the second step proceeded, either k3 > kÿ3 or the ligated exons (5E-3E) dissociate readily from the intron (k4 > kÿ3). The maturase itself binds tightly to the intron and turns over slowly (0.006 minÿ1; Ho et al., 1997). The overall rate of the second splicing step is about 1.5 minÿ1 (Ho et al., 1997). As the rate of the second step was increased by either decreasing the Mg2‡ concentration from 25 to

990 5 mM Mg2‡, increasing the temperature from 30 to 37  C, or truncating the 30 exon (removing vector sequence), it is possible that the sequence ¯anking the 30 SS is loosely sequestered in an inactive conformation that may not be present in vivo (data not shown). Only the ®rst step of splicing will be analyzed in the experiments described below. As k2 > kÿ2 < k3, we will assume that this step is irreversible to a ®rst approximation and that kobs  k2 under standard conditions. In self-splicing, the I3E intermediate accumulates to an approximately steady-state level of less than 3 % of total RNA (Hur et al., 1997; Ho et al., 1997). As exon ligation is much faster than the reverse of 50 splice-site cleavage in the Tetrahymena intron (Suh & Waring, 1993) where accumulation of the I3E intermediate is also minimal, kobs may approximate to the equivalent of k2 above (the forward catalytic step of the ®rst splicing reaction). However it currently remains possible that kobs for 50 SS self-cleavage is a function of several rate constants (the self-splicing equivalents of k2 through to k4 in Scheme 1). The maturase is not a chaperone The E. coli ribosomal S12 protein transiently assists group I intron splicing by acting as an RNA chaperone: the protein factor can be removed by protease K digestion after the RNA precursor is folded without preventing RNA splicing (Coetzee et al., 1994). Protease digestion experiments were carried out to determine whether maintenance of the RNA-maturase complex was required during splicing. The maturase was allowed to bind prefolded RNA precursor in 5 mM MgCl2 for ten minutes. Protease K was added and after 15 minutes incubation, the reaction was started with GTP. Protease K completely abolished protein-assisted splicing (Figure 2, lane 2) but did not inhibit RNA self-splicing per se (Figure 2, lane 4). The results indicate that maturase binding is required to maintain splicing activity at 5 mM Mg2‡ and that the maturase is not an RNA chaperone. The effect of pH on AnCOB intron splicing Generally, group I intron self-splicing shows a pH dependence in the acid range, and becomes pH independent at higher pH with a pKa of close to 7.0 (Herschlag & Khosla, 1994; Zaug et al., 1994; Weeks & Cech, 1995; SjoÈgren et al., 1997). These reports have suggested that the pH-independent phase represents a conformational step and have argued that a linear relationship between the pH and the log of the rate constant with slope of ‡1 indicates that the catalytic step involves deprotonation. While not expecting the AnCOB intron to deviate radically from this trend, we wished to know which of these steps was primarily determining the reaction rate under our standard conditions. The pH dependence of AnCOB splicing

Maturase-assisted Splicing of a Group I Intron

Figure 2. The maturase is not an RNA chaperone and is required during splicing. Lane 1, unreacted RNA precursor. Lane 2, 1 nM prefolded RNA precursor was allowed to bind 5 nM maturase (lot c) in 10 ml of splicing buffer for ten minutes at 37  C; 1 ml protease K was added (2 mg/ml ®nal) followed 15 minutes later by GTP. Lane 3, as described for lane 2 minus protease K. Lane 4, self-splicing reaction ‡2 mg/ml protease K. The arrow indicates a degradation product sometimes seen in gel-puri®ed pre-RNA. Reactions in lanes 2, 3 and 4 were incubated for 60, 3 and 60 minutes, respectively, after addition of GTP to 1 mM. Lanes 2 and 3 have less RNA, as the completed reactions were phenolized to remove protein prior to gel electrophoresis.

was determined in the presence and absence of the maturase. In self-splicing conditions and 25 mM Mg2‡, the ®rst-order rate constant for splicing increased with increasing pH in the acid range. Above pH 7, kobs became independent of pH (Figure 3). This behavior is similar to that obtained with other group I introns and the data were therefore ®tted to the appropriate equation (Figure 3). The pKa was 6.3 and the kmax,pH was 0.07 minÿ1. The experimental slope could not be determined under self-splicing conditions because only a few measurements were made below the pKa. The maturase-assisted splicing reactions showed a similar pH-dependent pro®le but with a pKa of 7.0, a kmax,pH of 11 minÿ1 and an observed slope of ‡1 in the acid range (Figure 3). A preliminary experiment using only Tris buffer and a different lot of maturase preparation gave a pKa of 7.0 and a kmax,pH of 7.2 minÿ1. The pro®les in Figure 3 show that our standard conditions (pH 7.4) primarily monitor the pH-independent step in the presence of the maturase and almost exclusively monitor this step under self-splicing conditions.

991

Maturase-assisted Splicing of a Group I Intron

Figure 3. pH dependence of self-splicing and maturase-assisted splicing. Protein-assisted splicing was initiated by addition of GTP to the prebound RNAmaturase complex in the buffers described in Materials and Methods. Triangles, self-splicing; squares, maturaseassisted splicing (lot c). The data were ®tted (Fersht, 1985) to kobs ˆ (kmax,pHKa)/([H‡] ‡ Ka).

The maturase is more proficient than Mg2‡ in assisting AnCOB splicing Divalent metal ions, Mg2‡ or Mn2‡, are required for group I intron splicing. Under single turnover conditions, the reaction rate of maturase-assisted splicing was insensitive to the Mg2‡ concentration from 4 to 100 mM (kobs ranged from 4.7 to 3.2 minÿ1, lot b, Figure 4). In contrast, under self-splicing conditions, in 1 mM GTP, kobs increased from 0.007 minÿ1 at 15 mM MgCl2 to a maximal rate of 0.38 minÿ1 at 150-200 mM MgCl2 (Figure 4). This was still only 10 % that of the maturase-assisted reactions, indicating that high Mg2‡ concentration alone is not able to substitute for the maturase. The results suggested that magnesium ions co-operate in the folding of the pre-RNA and so the self-splicing data were ®tted to the Hill equation (Figure 4). The ®t indicated that 69 mM Mg2‡ was required to achieve half the maximal rate seen with saturating Mg2‡ and yielded a Hill coef®cient of 2.4. The magnesium ions that co-operate in folding may not necessarily be located at discrete sites. The maturase-assisted reaction appeared to be inactive in 2 mM Mg2‡ and slower in 3 than 4 mM Mg2‡ (data not shown). However, reactions at low concentrations could have been affected by chelation of approximately 1 mM Mg2‡ upon addition of 1 mM GTP to start the reaction and so selected experiments were performed with guanosine (using maturase lot c). A reaction in 3 mM Mg2‡ and 0.2 mM guanosine (kobs ˆ 8.5 minÿ1) was similar to one in 5 mM Mg2‡ and 1 mM GTP (standard conditions) (kobs ˆ 8.0 minÿ1). In 2 mM Mg2‡ and

Figure 4. Saturating Mg2‡ stimulates RNA splicing less well than the maturase. Protein-assisted splicing was initiated by addition of GTP to the prebound RNAmaturase complex (see Materials and Methods). Two independent experiments were performed for self-splicing. Squares, self-splicing; triangles, maturase-assisted splicing (lot b). The primary data were analyzed as described in Materials and Methods. The self-splicing data were ®tted to the Hill equation: log[kobs/ (kmax,Mg ÿ kobs)] ˆ nlog[Mg2‡] ÿ log[Mg2‡]n1/2, where n is the Hill coef®cient. This gave kmax,Mg ˆ 0.36 minÿ1; n ˆ 2.4 and [Mg2‡]1/2 ˆ 69 mM.

0.2 mM guanosine, 33 % of the RNA was involved in a fast burst (kobs ˆ 6 minÿ1) with 90 % reacting overall. We conclude that the maturase-assisted reaction is Mg2‡ independent down to 3 mM and is still active in 2 mM Mg2‡. Dissociation of the RNA-maturase complex is slow Dissociation of labelled pre-RNA from the maturase was examined by measuring the stability of the complex in the presence of excess competitor, cold pre-RNA, using a nitrocellulose binding assay (data not shown). This gave a value for the dissociation rate constant, koff, of about 0.04 minÿ1. Using a nitrocellulose binding assay, the Kd had previously been measured to be 3 nM in 25 mM Mg2‡ at 28  C (Ho et al., 1997). A Kd of 2 nM was obtained when the assay was repeated at 37  C in 5 mM Mg2‡ (data not shown). This, together with an estimate of kon (see below), indicated that koff should be of the order of 6 minÿ1. Given the disparity in the nitrocellulose assays, dissociation was monitored by following the reaction of labelled pre-RNA complexed to protein in the presence of excess cold pre-RNA. As the protein appears to bind non-speci®cally to a variety of RNAs and DNAs under certain conditions (excess protein, RNA or DNA and depending on the ionic strength of the buffer, data not shown), the amount of cold pre-RNA was kept low. However this meant that there was insuf®cient cold RNA to ef®ciently prevent dissociated labelled RNA from rebinding (see

992 below). The maturase concentration was 3 nM and labelled and cold RNA were 0.25 nM and 10 nM, respectively. Three reactions were initiated with GTP (Figure 5). The ®rst was a standard reaction control. In the second, reaction of the prebound complex was initiated by simultaneous addition of GTP plus excess cold RNA. Its similarity to the ®rst indicates that virtually all the bound RNA reacted and that koff5k2 (where k2  kobs as per the analysis of Figure 1 and Scheme 1). The third reaction was a control. Labelled and cold competitor RNA were mixed and the reaction started by the simultaneous addition of GTP and maturase. Because the competitor concentration was suboptimal, this control revealed the minimal fraction of labelled RNA expected to react if the cold and labelled RNA rapidly equilibrated (koff4k2). Assuming all the protein (3 nM) was active and a 1:1 ratio of RNA to protein, 3 nM of a total of 10.25 nM RNA should have complexed

Maturase-assisted Splicing of a Group I Intron

with protein. Therefore 29 % of the labelled RNA was expected to react. In reality (Figure 5), 38 % reacted. The 30 SS of group I introns is susceptible to hydrolysis (Zaug et al., 1985). To get a better estimate of koff, the same reaction and two controls were repeated under hydrolysis conditions at pH 8.1 without GTP (Figure 5). The two controls set the two extreme reaction pro®les one might theoretically expect to see. The ``no competitor control'' reaction, initiated without competitor RNA, is analogous to a chase reaction in which all the prebound labelled RNA reacts without dissociating (koff5khyd). The second control, initiated by the addition of protein to a mixture of labelled and cold RNA, is analogous to a chase reaction in which the labelled prebound RNA rapidly dissociates and equilibrates with the cold chase RNA (koff4khyd) and on the basis of the equivalent GTP control above, 38 % of the labelled RNA should react. The actual chase experiment was much more similar to the no competitor control (Figure 5), indicating that koff < khyd and that more RNA stays bound and reacts than dissociates. At pH 8.1, khyd was 0.04 minÿ1 (Figure 5), indicating that koff 4 0.04 minÿ1. Both the GTP and hydrolysis reactions indicate that complex formation is essentially irreversible during splicing with every binding event leading to cleavage of the 50 SS rather than dissociation of the complex (k24koff in Scheme 1). Analysis of the maturase-assisted reaction including the binding step

Figure 5. Analysis of the dissociation of the RNAmaturase complex. Final concentrations were: maturase 3 nM, labelled RNA 0.25 nM and cold competitor RNA 10 nM. RNA was prefolded (see Materials and Methods). Filled symbols: splicing conditions (1 mM GTP, pH 7.4); open symbols: 30 SS hydrolysis conditions (no GTP, pH 8.1). The controls are in broken lines. Squares, RNA and maturase were allowed to prebind for ten minutes and then cold RNA was added ‡ GTP (or without GTP for the hydrolysis reaction; some preRNA was hydrolyzed during prebinding at pH 8.1). Triangles, control reactions were started by addition of maturase ‡ GTP (no GTP in the hydrolysis reaction) to labelled RNA with no cold RNA. Diamonds, as for the triangles, but addition was to a premixture of cold and labelled RNA. The chased hydrolysis reaction (open squares) was ®tted to a single exponential equation (see Materials and Methods) and yielded: 0.77eÿ0.03t ‡ 0.17. The hydrolysis control (open triangles) was ®tted to Fpre ˆ A[(1/(ky ÿ kx))(ky eÿkx t ÿ kx eÿky t )] ‡ (1 ÿ A) which describes two consecutive irreversible reactions. This gave kx and ky ˆ 0.21 and 0.04 minÿ1. The rate constant for hydrolysis, khyd, was assumed to be the lower value (0.04 minÿ1) given the slow rate (0.03 minÿ1) of the chase reaction. The other step may be a conformational change required before hydrolysis can take place.

All the experiments described thus far monitored the reaction subsequent to formation of the RNAprotein complex. The splicing factors Cbp2 and CYT-18 employ different mechanisms to stabilize the intron RNA (see the Introduction), but these differences are not apparent when analyzing prebound complexes. We also wished to determine if there was a rate-limiting step prior to attainment of the equilibrated state of the bimolecular complex. If such a step exists, it will limit the rate of 50 SS cleavage and can be measured under conditions where the maturase is present in such high concentrations that binding itself is not rate-limiting. Under such conditions, the reaction should be pseudo-®rst-order. Single turnover experiments were performed with varying concentrations of maturase (lot b) without prebinding. The primary data (obtained with higher concentrations of maturase) were ®tted to a biphasic exponential equation due to the presence of a much slower phase that was similar in size (10-15 %) and rate (0.1-0.2 minÿ1) to the one seen in the earlier experiments with prebound complex. The kobs value for the fast phase was plotted against maturase concentration (Figure 6, inset). The reaction reached a maximum rate (kmax) of 5 minÿ1. The reactions performed at 3-5 nM maturase were not ®tted to a biphasic exponential

993

Maturase-assisted Splicing of a Group I Intron

Figure 6. Measurement of the rate of the catalytic step for the reaction involving free RNA and maturase. A standard splicing reaction was performed with 0.5 nM prefolded RNA omitting the prebinding step: the reaction was started by simultaneous addition of maturase (lot b) and GTP. Maturase concentrations were 7.5 (open square), 10 (triangle), 20 (diamond), 30 (open triangle), 40 nM (cross). The reaction slowed noticeably below 10 nM maturase: a 4 nM reaction (circle), not ®tted, is included for comparison. The data were ®tted to a double exponential equation (see Materials and Methods and the text for further details) and kobs for the fast phase plotted against maturase concentration (inset).

curve because the reactions could not be assumed to be pseudo-®rst-order (the concentration of the protein contributes to the overall reaction rate and koff is so low that bound and free RNA are not readily in equilibrium). They were not included in Figure 6 but were analyzed separately (Figure 7). In most experiments, there was usually no signi®cant difference between reactions performed with or without a prebinding step providing the concentration of maturase was 510 nM. This was true for maturase lot b (kobs and kmax ˆ 4 and 5 minÿ1, respectively; compare Figures 4 and 6), maturase lot a (6.5 and 6.0 minÿ1, respectively) and lot c (11 and 11 minÿ1, respectively) (data not shown). These two reactions are therefore very likely to share the same rate-limiting step. Reactions performed without prebinding but with excess protein showed a similar accumulation of the I3E intermediate as seen in the reaction of the prebound complex (Figure 1(b)). In summary therefore, the data are consistent with the conclusion that kmax  k2  kobs, where kmax is derived from Figure 6, k2 is the rate constant for the step subsequent to binding from Scheme 1, and kobs represents the rate constant for the reaction of the prebound complex (Figure 1(b)). This means that the status of the prebound bimolecular complex is rapidly attained after binding of free RNA and protein without being delayed

Figure 7. Estimate of the association rate constant assuming that binding is irreversible. Standard reactions were performed without prebinding as outlined in the legend to Figure 6 but using lower concentrations of maturase. Individual reactions were ®tted to Scheme 2 describing two consecutive irreversible ®rst-order reactions (see Materials and Methods) to obtain the pseudo®rst-order rate constants [M]kon, where kon is secondorder association rate constant and [M] is maturase concentration. Open symbols and continuous lines, maturase lot b and 0.5 nM RNA; ®lled symbols and dotted lines, lot c and 0.25 nM RNA. Circles, 2; squares, 3; triangles, 4; diamonds, 5; and stars, 7.5 nM maturase. Inset: Squares, lot c; triangles, lot b. The derived value of kon ˆ 3  109 Mÿ1 minÿ1.

by an intervening slow folding step. The data also argue that the rate-limiting step is not a kinetically unfavorable RNA folding event which must be trapped by the protein as in the case of Cbp2, but one that occurs after binding as in the CYT-18 reaction (see the Discussion). The nature of the rate-limiting step is discussed later. Estimate of the association rate constant The preceding experiments have shown that k24koff (Scheme 1), which means that complex formation is essentially irreversible. Since k2 > kÿ2, 50 SS cleavage can now be simpli®ed to two irreversible consecutive reactions: ‰MŠkon

‡G

k2

5E-I-3E ÿ! 5E-I-3E
M ÿ! 5E
G-I-3E
M Scheme 2 The ®rst step is pseudo-®rst-order under single turnover conditions with maturase in excess. The second (catalytic) step has already been analyzed (Figure 6). It is also pseudo-®rst order when performed in saturating GTP. Scheme 2 permitted the second-order rate constant (kon) for the association of the binary complex to be determined.

994 Directly monitoring the binding step itself was complicated by two factors: (1) the reaction rates were at the limit of what could readily be measured by manual mixing and analyzing scanned autoradiograms; (2) the fast rate of association prevented us from analyzing the reaction under conditions where binding was clearly ratelimiting because the concentration of maturase could not be lowered suf®ciently while still preserving single turnover conditions (maturase in excess and a detectable amount of RNA). Using sub-maximal concentrations of maturase, experiments were performed in which the reaction was started by simultaneously adding GTP and maturase to prefolded RNA (Figure 7). The data were ®tted to an equation which describes Scheme 2 (see Materials and Methods; Fersht, 1985; Fierke & Hammes, 1995) to obtain the pseudo-®rst-order rate constant, [M]kon for each individual reaction. k2 in Scheme 2 was ®xed at kmax, the value obtained with saturating maturase (Figure 6; see Materials and Methods). The value of kon was estimate to be 3  109 Mÿ1 minÿ1 from the inset to Figure 7. Earlier time-points than ®ve seconds will be required to determine this more accurately. Combining estimates of the association and dissociation rate constants yielded an estimated Kd of 410 pM. Folding of the heat-denatured RNA is rate-limiting in the maturase-assisted reaction in vitro In the standard reaction the RNA was prefolded. The rate at which denatured RNA folded in the presence of the maturase was measured by heating the pre-RNA to 90  C, placing it on ice and adding it to a standard splicing buffer already containing maturase. In 5 mM Mg2‡, omitting the ®ve minute prefolding procedure introduced a rate-limiting step (k 5 1.3 minÿ1) which the maturase could not compensate for (Figure 8). This step could occur before or after maturase binding, but the former is more likely. In 25 mM Mg2‡, the reaction was slightly slower but went further to completion. While this analysis is required to fully investigate the in vitro reaction, it should be noted that the RNA will fold in vivo as it is synthesized and may also need to refold after being traversed by mitochondrial ribosomes. T1 RNase analysis of folding of AnCOB at different MgCl2 concentrations Ribonuclease T1 preferentially cleaves guanosine bases that are not involved in secondary or higherorder structure (Ehresmann et al., 1987). RNase T1 was used to monitor the folding of AnCOB in different concentrations of Mg2‡ or in the presence of the maturase (Figure 9). Guanosine bases were scored as being sensitive to T1 if they were cleaved to at least half the extent observed in the absence of Mg2‡. In summary: (1) addition of the maturase

Maturase-assisted Splicing of a Group I Intron

Figure 8. Folding of heat-denatured RNA is ratelimiting in the maturase-assisted reaction. RNA was heated and cooled as described in Materials and Methods. Final RNA and maturase (lot b) concentrations were 1 and 10 nM, respectively. Data were ®tted to either a single or double exponential equation. Filled squares, a standard reaction in which RNA was prefolded for ®ve minutes but without prebinding of RNA and maturase (Fpre ˆ 0.80eÿ3.72t ‡ 0.14eÿ0.28t ‡ 0.06); Open squares, as ®lled squares but with no RNA prefolding step (Fpre ˆ 0.76eÿ1.32t ‡ 0.16eÿ0.12t ‡ 0.08); Open triangles, as open squares but with 25 mM Mg2‡ (Fpre ˆ 0.92eÿ0.87t ‡ 0.08). The last two reactions were started by adding denatured RNA to standard splicing buffer containing maturase.

led to protection of all except three G bases, including several that were still partially sensitive in its absence in high concentrations of Mg2‡; (2) the maturase probably binds an RNA that has limited stable tertiary structure and even some unstable secondary structure. The pre-RNA without maturase was analyzed in the absence of Mg2‡ (semi-denaturing conditions), 2 mM Mg2‡ (the lowest concentration which effectively supports the protein-assisted reaction), 5 mM (standard conditions), 25 mM (which supports a low rate of self-splicing) and higher concentrations. Most of the G residues within the P4, P5, P5ab, P6, P6a and P8 regions were signi®cantly protected at 2 and even 0 mM Mg2‡. The G residues in the purine-rich region between P6 and P6a were well protected. G bases within P7, P9 and P9.1 were still sensitive to T1 in 5 mM Mg2‡ (Figure 9). P7 and P3 form late in the Tetrahymena intron folding pathway (Zarrinkar & Williamson, 1994, 1996; Banarjee & Turner, 1995; Sclavi et al., 1998) and are the least stable pairings in the core structure at equilibrium in low Mg2‡ (Celander & Cech, 1991; Laggerbauer et al., 1994). In 2 and even 5 mM Mg2‡, several G bases in single-stranded regions were as sensitive as those within the 50 exon, implying they were primarily unfolded. A number were still sensitive to T1 in 25 mM Mg2‡ (Figure 9) and several were partially sensitive in up to 100 mM Mg2‡ (data not shown), but scored as protected compared to 0 Mg2‡.

Maturase-assisted Splicing of a Group I Intron

The ®rst G base (G248) of the GNRA L9 loop was more sensitive to T1 in the absence of the maturase than the equivalent G25 in the GNRA L2 loop. (In GNRA loops, this G base usually forms a sheared base-pair with the A base (Heus & Pardi, 1991; Pley et al., 1994) or an asymmetric heteropurine (Perbandt et al., 1998)). This may be because the P9 stem is extremely A ‡ U-rich and less stable than P2 or due to the difference in loop sequences (GUGA and GAAA). In summary, the protection data indicate that the paired regions from P4 to P6 are the most stable in AnCOB and that the P7 and (possibly) the P3 helices are unstable at low concentrations of Mg2‡. Protection by the maturase In the presence of the maturase at 5 mM Mg2‡, all guanosine bases except three (G105, G122, G215) were resistant to T1 cleavage. Particularly noticeable was the resistance of G bases ¯anking P9.1, the G bases lying between P5 and P5ai and G9 in L1, most of which are not believed to be involved in Watson-Crick base-pairs. These G bases remained partially sensitive in high concentrations of Mg2‡ alone (see above). This demonstrates that the maturase stabilizes the tertiary structure of the intron. There is strong phylogenetic and experimental evidence in other introns to believe that G27 and A28 in L2 are part of a GNRA loop that forms a tertiary interaction with A221 and G222 of P8 (Michel & Westhof, 1990; Jaeger et al., 1994; Costa & Michel, 1995, 1997; Hur et al., 1997). G27 was well protected in the presence of the maturase (Figure 9). In contrast, protection of G27 was poor in 25 mM Mg2‡ alone (Figure 9) and only partial in 50-100 mM Mg2‡ (data not shown). The 50 SS is located in P1 formed by base-pairing between the 50 exon and the internal guide sequence (IGS) (Figure 9(b)). G12 in the IGS and the 50 exon G residues G ÿ 4 and G ÿ 5 were sensitive to T1 in low Mg2‡ concentrations, but protected in the presence of the maturase, indicating that P1 is unstable in low Mg2‡ concentrations. The 50 exon residues G ÿ 7, G ÿ 8 and G ÿ 10, upstream of P1, became hypersensitive to T1 RNase in either 5 mM Mg2‡ plus maturase or 525 mM Mg2‡ alone, suggesting that these nucleotides may contribute directly or indirectly to the instability or misfolding of P1 in low Mg2‡ concentration (Figure 9).

Discussion The maturase is clearly an excellent splicing factor for the AnCOB group I intron. It dramatically accelerates 50 SS cleavage, even at 2 mM Mg2‡. Kinetic analysis and T1 RNase protection assays showed that it binds tightly to the intron RNA. Sensitivity of the reaction to proteinase K indicates that the protein is not a chaperone and that it must

995 be present during the reaction. The vulnerability of the intron to T1 RNase cleavage in the absence of the maturase in 45 mM Mg2‡ argues that it stabilizes RNA tertiary structure and some secondary structure. It is possible that the T1 RNase was primarily cleaving signi®cantly misfolded RNA molecules that were slow-reacting or inactive (about 15 %) rather than active molecules at some stage of the equilibrium folding pathway. Several observations argue against this: there was virtually no residual cleavage of most protected G bases in the presence of the maturase. Native gels (as performed by Caprara & Waring, 1993) did not reveal more than one species of molecule at 10 nM RNA (data not shown). Some G residues were as sensitive in 5 as in 0 mM Mg2‡ and, while tRNA tertiary structure is stable though slowly attained without Mg2‡ (Cole et al., 1972; Draper, 1996), unmodi®ed tRNAs (Maglott et al., 1998) and pro®cient self-splicing introns have limited stable tertiary structure in 0 mM Mg2‡ and 0.1 M monovalent salt (Celander & Cech, 1991; Downs & Cech, 1996). Some of the unpaired G bases were as sensitive as those in the 50 exon well upstream of P1 (Figure 9(a)). These observations argue that the T1 RNase was predominantly cleaving true native folding intermediates rather than misfolded non-native structures. This work provides an overview of the kinetics of maturase-assisted splicing. The unexpectedly fast rate of both binding and catalysis made it dif®cult to accurately measure the values of the kinetic constants. However this analysis should provide the initial framework needed to determine these more accurately and to study mutants. Both self-splicing and maturase-assisted splicing were pH dependent in the acid range with a pKa of 6.3 and 7.0, respectively. The self-splicing reactions of other group I introns have a pKa of close to 7.0. These introns are generally more stable than AnCOB. At 25 mM Mg2‡, AnCOB is unstable (Figure 9) and so it is likely that the pH-independent rate-limiting step is acquisition of the fully folded tertiary structure. Because this is so slow in 25 mM Mg2‡ (0.07 minÿ1), a signi®cant drop in pH is necessary before the chemical step becomes ratelimiting. For most introns, it has been suggested that the pH-independent step is an unidenti®ed conformational change (Herschlag & Khosla, 1994; Zaug et al., 1994; Weeks & Cech, 1995; SjoÈgren et al., 1997). The pH pro®le of the maturase-assisted reaction was similar to that of pro®cient self-splicing introns. The native maturase is likely to have a pI that is similar to the value of 10 calculated for the bacterially expressed protein. Although titration of multiple non-basic amino acids might have some minor effect, the pH pro®le below pH 7 is most likely explained by a chemical step involving loss of a proton. The nature of the pH-independent rate-limiting step is unknown. As the pH of the interior of the mitochondria is probably slightly basic, deprotonation is unlikely to be the major

996

Maturase-assisted Splicing of a Group I Intron

Figure 9 (legend shown on opposite page)

rate-limiting step in vivo, unless the pH-independent step is signi®cantly enhanced. Dissociation of the maturase-RNA complex (Figure 5) is much slower (40.04 minÿ1) than catalysis (11 minÿ1) arguing that binding is essentially irreversible with every RNA that binds reacting rather than dissociating. Slow dissociation of the precursor and maturase is consistent with slow release of the splicing products under multiple turnover conditions (0.006 minÿ1; Ho et al., 1997). Binding of the maturase is fast. The secondorder rate constant (kon) is estimated to be about 3  109 Mÿ1 minÿ1. This is nearly as fast as the binding of tRNAs to their synthetases (kon ˆ 1  1010 Mÿ1 minÿ1, Fersht, 1985). Combining estimates of the association and dissociation rate constants provides an estimate of the Kd at 410 pM. It should be noted that kon and koff were estimated in the presence and absence of GTP, respectively, and that GTP could contribute to

either. The reason that a nitrocellulose binding assay yielded such a high Kd value is unknown; varying the length of time for the binding reactions con®rmed that they did reach equilibrium. Two other group I intron splicing factors have been well characterized: Cbp2 assists the ScCOBi5 intron from Saccharomyces cerevisiae (Gampel et al., 1989) and CYT-18 assists the LSU rRNA and other introns from N. crassa (Akins & Lambowitz, 1987). CYT-18 nucleates a poorly folded intron, establishing and drawing together two major helical domains to form the core structure (Caprara et al., 1996; Saldanha et al., 1996). It has been proposed that Cbp2 acts by trapping a rate-limiting tertiary conformational change in the RNA so that the reaction rate is independent of protein concentration (Weeks & Cech, 1996). (It has also been argued that Cbp2 assists formation of the intron secondary structure (Lewin et al., 1995)). Nevertheless, the actual binding step involving Cbp2 has a similar

Maturase-assisted Splicing of a Group I Intron

997

Figure 9. The maturase stabilizes the AnCOB intron and protects it from T1 RNase. (a) Primer extension of T1 RNase cleaved pre-RNA; 0 mM Mg2‡ included 50 mM EDTA; 50 nM bovine serum albumin (BSA) was used instead of maturase as a control. The No T1 lane was treated as for the 5 mM Mg2‡ reaction but without T1. Two bands above G9 were due to nonspeci®c termination by reverse transcriptase. *G-7, -8 and -10 upstream of P1 were hypersensitive to T1 in high Mg2‡ or ‡ maturase. Selected G bases are numbered. Px and Px0 ˆ 50 and 30 portion of a paired stem, respectively. (b) G bases were scored as being sensitive to T1 if cleavage was 550% the level seen in 0 mM Mg2‡. The analysis represents the average of three experiments. G bases that became protected by the presence of the maturase in 5 mM Mg2‡ are shown in bold lower-case. Pairing P10 between the 30 exon and the internal guide sequence is indicated by thin lines. The 50 exon nucleotides are numbered negatively. Double-arrowed lines indicate likely tertiary interactions between the boxed regions: L2/P8 and L9/ P5 (Michel & Westhof, 1990). G295, G313 and G ‡ 5 (in 30 exon) were not scored.

association rate constant (3  109 Mÿ1 minÿ1; Weeks & Cech, 1996) to that of the maturase and CYT-18 (2  109 Mÿ1 minÿ1; Saldanha et al., 1995). In principle, kmax for the reaction involving free RNA and maturase (Figure 6) could represent either a protein-independent, RNA folding step before binding (Cbp2-like), or a step after binding (CYT-18-like). The latter is much more likely because the reaction rate was dependent on the maturase concentration until it reached the rate measured for the prebound binary complex. The maturase therefore appears to behave more like CYT-18. Compared to the other two protein-assisted reactions, the catalytic step is unusually fast. The CYT18 and Cbp2 assisted reactions have rate constants of about 0.2 and 1 minÿ1, respectively, for prebound complexes (Saldanha et al., 1995; Weeks & Cech, 1996) compared to 11 minÿ1 for the maturase reaction. This is faster than all the self-splicing reactions examined, except for the Anabaena intron

where a kcat of 14 minÿ1 has been measured (Zaug et al., 1994). What is rate-limiting in the catalytic step of 50 SS cleavage? Whether the maturase changes the ratelimiting step compared to ef®cient self-splicing introns or whether it simply increases the rate of a common limiting step is unknown as yet. At pH 7.4 the step is almost pH independent (Figure 3) so it is unlikely to include chemistry. It is Mg2‡ concentration independent (Figure 4) implying that the observed rate-limiting step is not binding of an essential Mg2‡. The kobs value under standard conditions was similar at 30 and 37  C, 25 % slower at 28  C and much slower at 25  C (data not shown). While this might suggest that the rate-limiting step is relatively insensitive to temperature, given the complexity of the splicing reaction, it is also possible that opposing compensatory effects offset one another within a range of 10 deg. C. The kmax value can be attained without prior complex formation under pseudo-®rst-order

998 conditions at 510 nM maturase, therefore the ratelimiting step cannot represent a thermodynamically favorable, but slow, conformational change (or folding step) subsequent to RNA-maturase binding but prior to binding of GTP. An unfavorable conformational change prior to GTP binding remains a possibility. A rapid post-binding conformational change (k ˆ 30 minÿ1) involving the LSU intron and CYT-18 has been detected using stopped-¯ow methods (Saldanha et al., 1995). Other possibilities include a conformational change in RNA and/or protein after GTP binding and just prior to cleavage of the 50 SS. What does the maturase accomplish to stimulate such a high rate of splicing? It clearly shifts the equilibrium of the folding pathway beyond what can be achieved by optimal levels of Mg2‡ alone. In doing so, it directly or indirectly facilitates binding of the Mg2‡ involved in catalysis, establishment of the guanosine cofactor binding site and docking, and possibly folding (Figure 9) of the P1 substrate. The maturase may also suppress the large-scale thermal motion observed in larger RNAs (Cohen & Cech, 1997). At 25 mM Mg2‡, certain G nucleotides are partially sensitive to T1 but there is also limited self-splicing activity (kobs ˆ 10 % of the maximal rate in high Mg2‡ and 1 % of the maturase-assisted reaction). Some of these G bases are almost undoubtedly involved in tertiary interactions (e.g. G27), arguing that at 25 mM Mg2‡ the intron is in equilibrium between a fully and partially folded state with the latter greatly predominating. In 5 and especially 2 mM Mg2‡, there is further destabilization of tertiary structure and partial destabilization of secondary structure at P7, (P3?), P9, and P1 (Figure 9), and yet the maturase readily activates the RNA at 2 mM Mg2‡. To what degree then does the maturase recognize an unfolded RNA in 45 mM Mg2‡? Although folding equilibria may be displaced temporarily in favor of more folded states at any instant, the kinetic data argue against a pronounced trapping mechanism (see above). Overall we believe that the maturase behaves like CYT-18 in that it recognizes a partially unfolded RNA and then rapidly establishes a stable, highly active conformation. A thorough analysis of the intron's accessibility to chemicals used to probe RNA structure will clarify the situation. Although the maturase may be able to correct misfolded structures, this cannot be its sole function because it is required during the splicing reaction (Figure 2). Therefore the maturase is not a true chaperone. However, it can be described as having chaperone activity, since it helps to fold the intron RNA (Figure 9). (For a discussion of this distinction, see Herschlag, 1995.) Heat-denatured RNA reacts more slowly than prefolded RNA (Figure 8), arguing that there is an early step in folding that cannot be as readily (if at all) assisted by the maturase. The Tetrahymena P1 helix, containing the 50 splice-site, is assisted by the co-axial hairpins P2/

Maturase-assisted Splicing of a Group I Intron

L2 and P2.1/L2.1 which form tertiary interactions with L5c and L9.1 respectively (Lehnert et al., 1996). This results in P1 being orientated at rightangles to P2 and P2.1. In contrast, in the T4 td intron (subgroup IA2) the L2 loop formed by P2 makes a tertiary interaction with P8 (Michel & Westhof, 1990; Costa & Michel, 1995, 1997). As L2 is a ®xed distance from P1 in such introns, the authors have argued that the L2/P8 interaction helps P1 dock to the core structure and that P1 and P2 are co-axial. Phylogenetic analysis argues that there is a similar L2/P8 interaction in subgroup IB4 introns like AnCOB (Michel & Westhof, 1990; Hur et al., 1997). The T1 ribonuclease assays are consistent with this model and also suggest that the maturase corrects misfolding of the P1 helix. Why is splicing of AnCOB so dependent on a protein factor when many other group I introns self-splice so ef®ciently? A simple explanation is that the intron is A ‡ U-rich and thus less able to form a stable and active tertiary structure (and even secondary structure in the case of the A ‡ Urich P9; Figure 9(b)). As large and even small RNAs are prone to becoming permanently or temporarily misfolded (Down & Cech 1996; Pan & Woodson, 1998; Esteban et al., 1997; Stage-Zimmerman & Uhlenbeck, 1998), there may also have been selection against mutated AnCOB sequences which folded more independently but misfolded more frequently. All known maturases are expressed from intronic ORFs fused to the upstream exon; in contrast, homing endonucleases can be free-standing (Lambowitz & Perlman, 1990; Belfort & Roberts, 1997). This arrangement ensures that expression of the maturase is subject to negative feed-back regulation, since intron excision prevents ORF translation (Lazowska et al., 1980). It is not known if this mode of regulation is obligatory. The tight binding of the AnCOB maturase and its ability to facilitate rapid RNA folding suggest that the maturase may counter the disruptive passage of ribosomes by either hindering translocation or rapidly refolding the RNA.

Materials and Methods Expression and purification of the maturase This was as described (Ho et al., 1997). Protein concentration was determined by the Bradford assay and protein activity checked by measuring the size of the burst under multiple turnover conditions (Ho et al., 1997). RNA precursor synthesis Uniformally labelled RNA precursors were transcribed from pCOBsal (Hur et al., 1997) as described (Suh & Waring, 1993). The AnCOB precursor generated from pCOBsal linearized with PvuII contains 112 and 209 nt of 50 and 30 exon, respectively, of which 97 and 26 nt are from the cobA gene. The intron is only 311 nt because 753 nt were deleted from L8 and replaced with the sequence UGUCGACAUA containing a SalI site

999

Maturase-assisted Splicing of a Group I Intron (Figure 9(b)), the deletion has little effect under conditions tested (Hur et al., 1997). In this work, AnCOB will refer to this AnCOB-L8 construct. Precursor RNA was puri®ed on a denaturing polyacrylamide gel (Suh & Waring, 1993). RNA concentration was determined using a scintillation counter. Standard splicing reactions Maturase-assisted, single turnover conditions The maturase was freshly diluted into 50 mM Tris (pH 7.4), 0.1 M KCl and added at less than one eighth ®nal volume. Precursor RNA was heated to 90  C in 10 mM Tris (pH 7.4), 0.1 mM EDTA for one minute and prefolded in splicing buffer (50 mM Tris (pH 7.4), 0.1 M KCl and 5 mM MgCl2) for ®ve minutes at 37  C. RNA (0.25 - 1 nM) was allowed to prebind with 10 nM maturase (usually at least tenfold excess) for ®ve minutes at 37  C and the reaction started by adding 1 mM GTP (all concentrations are ®nal). Reactions were stopped with phenol and EDTA (100 mM ®nal), organically extracted and analyzed on a denaturing 6 % polyacrylamide gel. Reaction pro®les were independent of RNA concentration (0.25 to 1.5 nM, data not shown) con®rming that they were being performed under single turnover conditions. Under standard conditions, which includes prebinding of RNA to the maturase, there was no signi®cant change in the reaction pro®le if the maturase concentration was doubled to 20 nM, indicating that the RNA was saturated (data not shown). Analysis of a range of GTP concentrations con®rmed that 1 mM GTP was also saturating. Self-splicing This was performed as above using 1 nM RNA prefolded for ®ve minutes but without protein and with 25 mM Mg2‡ as standard conditions. Increasing prefolding up to 60 minutes did not increase kobs. Kinetic analysis The rate of the ®rst splicing step was estimated from reactions performed under single turnover conditions. An autoradiogram was quanti®ed by laser densitometry (Molecular Dynamics). Using the Marquardt-Levenberg algorithm and non-linear regression analysis (PSI-plot), the maturase-assisted data were ®tted to both a single and double exponential: Fpre ˆ Aeÿkt ‡ …1 ÿ A† and: Fpre ˆ A1 eÿk1 t ‡ A2 eÿk2 t ‡ …1 ÿ A1 ÿ A2 † where Fpre is the fraction of precursor remaining and (1 ÿ A) and (1 ÿ A1 ÿ A2) represent the fraction of unreacted RNA for the single and double exponential equations, respectively. A1 and A2 represent the size of the fast and slow phases respectively. k1 and k2 represent the rate constants (minÿ1) of the fast and slow phases respectively. Generally, the reactions (standard conditions) showed a better ®t to a double exponential. The self-splicing reactions were ®tted to a single exponential equation (see above).

kmax rather than kcat is used to describe the rate constant for the catalytic step (equivalent to k2 in Schemes 1 and 2) because the reactions were not performed under steady-state conditions and there is no evidence that the protein plays a truly catalytic role in the reaction. In Figure 7 the data were ®tted to an equation which describes two irreversible reactions (see Scheme 2) (Fierke & Hammes, 1995), but also included a single exponential term (Beÿk3 t ) to represent a >20-fold slower phase: Fpre ˆA…1=…k2 ÿ ‰MŠkon ††…k2 eÿ‰MŠkon t ÿ ‰MŠkon eÿk2 t † ‡ Bek3 t ‡ …1 ÿ A ÿ B† where [M]kon is the apparent ®rst-order rate constant for complex formation under single turnover conditions and kon is the second-order association rate constant. k2 was ®xed at the value of kmax determined as described (Figure 6: for lots b and c, kmax ˆ 5 and 11 minÿ1, respectively). B was ®xed at 0.2 (the average size of the slow phase in the experiments performed). The values of A, [M]kon and k3 were obtained from the ®t. These ®ts were also performed with B allowed to ¯oat; the values of [M]kon varied up to 25 % for two individual reactions but the ®tted, derived value of kon was still 3  109 Mÿ1 minÿ1 (Figure 7, inset). pH pH dependence was determined under standard splicing conditions above except for the use of the following buffers: Mes, pH 5.8, and 6.3; Pipes, pH 6.3 (self-splicing only), 6.7; Tris, pH 6.7 (self-splicing only), 7.4, 8.1; and Ches, pH 8.7. Self-splicing reactions were usually slightly slower in Tris. Maturase-assisted reactions (1 nM RNA and 10 nM maturase (lot c)) were started by addition of GTP to the prebound RNA-maturase complex. The pH of the splicing buffers (1) was measured at 37  C. RNA was prefolded in splicing buffer. Above pH 9, RNA hydrolysis became signi®cant under self-splicing conditions. Mg2‡ dependence This was determined under standard splicing conditions above, except that for self-splicing the RNA was allowed to prefold for ten minutes. Maturase-assisted reactions 1 nM RNA and 10 nM maturase (lot b)) were started by addition of GTP to the prebound RNAmaturase complex. The self-splicing and maturaseassisted data were ®tted to single and double exponential equations, respectively (see above). In both situations, in >100 mM MgCl2 the fraction of unreactive precursor increased, presumably because more RNA was trapped in a mis-folded structure. T1 ribonuclease Precursor RNA was heated at 90  C for one minute and placed on ice immediately, 10 nM RNA was folded in 10 mM Hepes (pH 7.5), 100 mM KCl, at various MgCl2 concentrations at 37  C for ten minutes. A 1 mg sample of yeast tRNA (Sigma) was added after the preRNA was folded. Where required, 50 nM maturase (20 to 100 nM gave similar results) was added and incubated for ten minutes. The reactions (20 ml ®nal volume) were initiated by the addition of 0.001 unit of

1000 ribonuclease T1 (Pharmacia), incubated for four minutes at 37  C, terminated with 1 ml of 200 mM EDTA and extracted with phenol/chloroform, followed immediately by ethanol precipitation. Sites of ribonuclease T1 cleavage were mapped by reverse transcription (Caprara et al., 1996), using 50 labelled primers hybridized to either the 30 exon or L8 regions.

Acknowledgments Supported by a Grants-in-Aid award from Temple University. We thank William Geese for his assistance and Mark Caprara, Carol Fierke, Dan Herschlag, Ya-Ming Hou and Nicholas Macri for their helpful advice.

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Edited by D. E. Draper (Received 30 April 1999; received in revised form 22 July 1999; accepted 26 July 1999)