Self-splicing Reaction Research Articles

Probing the Tetrahymena group I ribozyme reaction in both directions.

The Tetrahymena L-21 ScaI ribozyme derived from the self-splicing group I intron catalyzes a reversible reaction analogous to the first step of self-splicing: CCCUCUA (S) + [UC]G right harpoon over left harpoon CCCUCU (P) + [UC]GA. To relate our understanding of the ribozyme to the self-splicing reaction and to further the mechanistic dissection of the ribozyme reaction, we have established a quantitative kinetic and thermodynamic framework for the forward and reverse reaction of the L-21 ScaI ribozyme under identical conditions. Examination of the framework shows that binding of products is cooperative with binding enhanced 5-fold, as was observed previously for binding of the substrates. Further, binding of UCGA is 12-fold weaker than binding of the unphosphorylated UCG, analogous to the 20-fold weaker binding of phosphorylated S relative to P; the molecular interactions underlying the stronger binding of UCG were traced to the 3'-hydroxyl group of UCG. The symmetrical effects on binding of substrates and products result in the equilibrium between ribozyme-bound species, K(int), that is essentially unperturbed from the solution equilibrium, K(ext) (K(int) = [E.P.UCGA]/[E.S.UCG] = 4.6 and K(ext) = [P][UCGA]/[S][UCG] = 1.9). Last, we show that the pK(a) values of the nucleophiles in the forward and reverse reactions are >/=10. This observation suggests that metal ion activation of the nucleophile and stabilization of the leaving group can only account for a portion of the rate enhancement of this ribozyme. These and prior results suggest that the Tetrahymena group I ribozyme, analogous to protein enzymes, uses multiple catalytic strategies to achieve its large rate enhancement.

Identification of an active site ligand for a group I ribozyme catalytic metal ion.

The transition state of the group I intron self-splicing reaction is stabilized by three metal ions. The functional groups within the intron substrates (guanosine and an oligoribonucleotide mimic of the 5'-exon) that coordinate these metal ions have been systematically defined through a series of metal ion specificity switch experiments. In contrast, the catalytic metal ligands within the ribozyme active site are unknown. In an effort to identify them, stereospecific (R(P) or S(P)) single-site phosphorothioate substitutions were introduced at five phosphates predicted to be in the vicinity of the catalytic center (A207, C208, A304, U305, and A306) within the Tetrahymena intron. Of the 10 ribozymes that were studied, four phosphorothioate substitutions (A207 S(P), C208 S(P), A306 R(P), and A306 S(P)) exhibited a significant reduction in the cleavage rate. Only the effect of the C208 S(P) phosphorothioate substitution could be significantly rescued by the addition of a thiophilic metal ion, either Mn(2+) or Zn(2+), when tested with an all-oxy substrate. The effect was not rescued with Cd(2+). To determine if one of the catalytic metal ions is coordinated to the C208 pro-S(P) oxygen, the phosphorothioate-substituted ribozymes were also assayed using oligonucleotide substrates with a 3'-phosphorothiolate or an S(P) phosphorothioate substitution at the scissile phosphate. This resulted in a second metal specificity switch, in that Mn(2+) or Zn(2+) no longer rescued the C208 S(P) ribozyme, but Cd(2+) provided efficient rescue in the context of either sulfur-containing substrate. The 3'-oxygen and the pro-S(P) oxygen of the scissile phosphate are both known to coordinate the same metal ion, M(A), which stabilizes the negative charge on the leaving group 3'-oxygen in the transition state. Taken together, these data suggest that metal M(A) is coordinated to the C208 pro-S(P) phosphate oxygen, which constitutes the first functional link between a specific catalytic metal ion and a particular functional group within the group I ribozyme active site.

Conserved Base-Pairings between C266–A268 and U307–G309 in the P7 of the Tetrahymena Ribozyme Is Nonessential for the in Vitro Self-Splicing Reaction

P7 is highly conserved in Group I self-splicing intron ribozymes. This region is known to coordinate metal ions and bind a cofactor guanosine required for the self-splicing. To further investigate the fundamental role of the corresponding region in the Tetrahymena ribozyme, we attempted to identify minimal requirements for the base-paired region excluding the guanosine binding site. We discovered that a variety of sequences are eligible and its derivatives possessing extra nucleotide(s) can still conduct the first step of splicing, indicating that no particular base-pairing is essential in this region for conducting the reaction in vitro. The results provide two hypotheses for the fundamental role of this region: (i) if the region contains element(s) that are strictly required in the catalysis, they are not necessarily tightly fixed in the ribozyme and (ii) if not, its fundamental role may simply be to coordinate neighboring regions that are directly involved in the catalysis.

Self-splicing of the Tetrahymena group I ribozyme without conserved base-triples.

Group I introns share a conserved core region consisting of two domains, P8-P3-P7 and P4-P6, joined by four base-triples. We showed previously that the T4 td intron can perform phosphoester transfer reactions at two splice sites in the absence of both P4-P6 and the conserved base-triples, whereas it is barely able to perform the intact splicing reaction due to the difficulty of conducting the sequential reactions. Based on previous findings, we constructed a bimolecular ribozyme lacking a large portion of P4-P6 and the base-triples from the Tetrahymena intron, on the assumption that the long-range interactions of the peripheral regions in the two RNAs can compensate for the deteriorated core. The bimolecular ribozyme performed the intact splicing reaction. The present analysis indicates that the base-triples are nonessential, but that L4 and the distal part of P4 in P4-P6 are important for conducting the splicing reaction. The reconstituted self-splicing ribozyme provides an amenable system for analysing the role(s) of elements in the core region in the self-splicing reaction mechanism.

Analysis of the P7 region within the catalytic core of the Tetrahymena ribozyme by employing in vitro selection.

The highly conserved P7 region is generally believed to act as a major portion of the catalytic site in the Group I intron ribozyme. However, its functions have not been elucidated except for the fact that it specifically binds a cofactor guanosine required for self-splicing reaction. We attempted an in vitro selection experiment to determine the sequence requirements of this region in the mechanism of catalysis by using the Tetrahymena ribozyme. We found that the selected active clones have the secondary structure similar to that of the wild type with few exceptions. However, their primary sequences were not conserved except G264 and C311 that are the major elements of the binding site for the guanosine. Our results suggest that the unique secondary structure of the P7 region is a primary requisite for the catalytic function of this class of ribozymes.

K + and Mg 2+ ions promote the self-splicing of the td intron RNA inhibited by spectinomycin

The effects of Mg 2+ and K + ions on the self-splicing inhibition of the td (thymidylate synthase gene) intron RNA by spectinomycin were investigated. The maximum splicing activity occurred at 20 mM KCl. The K m and V max values for GTP in the presence of 5 mM Mg 2+ are 2.25 μM and 0.55 min −1, whereas those for GTP both in the presence of 5 mM Mg 2+ and 5 mM K + are 1.23 μM and 0.46 min −1, respectively. Spectinomycin at 10 mM concentration inhibited the splicing by about 10%, but at 20 mM concentration, the splicing rate was inhibited by about 63%. The splicing inhibition by the low concentration of spectinomycin was overcome markedly as the concentration of Mg 2+ ion was raised. At 30 mM spectinomycin, however, the splicing inhibition was not significantly affected by increasing the concentration of Mg 2+. A similar activation of the splicing rate was observed as the concentration of K + ion was increased. The concentration of K + ion required for the normal recovery of the splicing was much higher than that of Mg 2+ ion. Unlike Mg 2+ ion, 30 mM K + ion effectively alleviated the splicing inhibition by spectinomycin at its high concentration. The results indicate that K + and Mg 2+ ions may show mechanistically different interactions with spectinomycin in the self-splicing reaction of the td intron RNA.

In vitro suicide inhibition of self-splicing of a group I intron from Pneumocystis carinii by an N3' --> P5' phosphoramidate hexanucleotide.

Binding enhancement by tertiary interactions is a strategy that takes advantage of the higher order folding of functionally important RNAs to bind short nucleic acid-based compounds tightly and more specifically than possible by simple base pairing. For example, tertiary interactions enhance binding of specific hexamers to a group I intron ribozyme from the opportunistic pathogen Pneumocystis carinii by 1,000- to 100,000-fold relative to binding by only base pairing. One such hexamer, d(AnTnGnAnCn)rU, contains an N3' --> P5' phosphoramidate deoxysugar-phosphate backbone (n) that is resistant to chemical and enzymatic decay. Here, it is shown that this hexamer is also a suicide inhibitor of the intron's self-splicing reaction in vitro. The hexamer is ligated in trans to the 3' exon of the precursor, producing dead-end products. At 4 mM Mg2+, the fraction of trans-spliced product is greater than normally spliced product at hexamer concentrations as low as 200 nM. This provides an additional level of specificity for compounds that can exploit the catalytic potential of complexes with RNA targets.

Group II intron splicing in chloroplasts: identificationof mutations determining intron stability and fate of exon RNA.

In order to investigate in vivo splicing of group II introns in chloroplasts, we previously have integrated the mitochondrial intron rI1 from the green alga Scenedesmus obliquus into the Chlamydomonas chloroplast tscA gene. This construct allows a functional analysis of conserved intron sequences in vivo, since intron rI1 is correctly spliced in chloroplasts. Using site-directed mutagenesis, deletions of the conserved intron domains V and VI were performed. In another set of experiments, each possible substitution of the strictly conserved first intron nucleotide G1 was generated, as well as each possible single and double mutation of the tertiary base pairing gamma-gamma ' involved in the formation of the intron's tertiary RNA structure. In most cases, the intron mutations showed the same effect on in vivo intron splicing efficiency as they did on the in vitro self-splicing reaction, since catalytic activity is provided by the intron RNA itself. In vivo, all mutations have additional effects on the chimeric tscA -rI1 RNA, most probably due to the role played by trans -acting factors in intron processing. Substitutions of the gamma-gamma ' base pair lead to an accumulation of excised intron RNA, since intron stability is increased. In sharp contrast to autocatalytic splicing, all point mutations result in a complete loss of exon RNA, although the spliced intron accumulates to high levels. Intron degradation and exon ligation only occur in double mutants with restored base pairing between the gamma and gamma' sites. Therefore, we conclude that intron degradation, as well as the ligation of exon-exon molecules, depends on the tertiary intron structure. Furthermore, our data suggest that intron excision proceeds in vivo independent of ligation of exon-exon molecules.

Identification of the nucleotides in the A-rich bulge of the Tetrahymena ribozyme responsible for an efficient self-splicing reaction.

P5abc is a large extension of the P5 element characteristic of subclasses IC1 and IC2 of group I introns. It has a conserved region termed the A-rich bulge, that is responsible for activation of the Tetrahymena self-splicing intron. By employing a modified color-colony assay system, we identified four adenosines in the bulge that are responsible for an efficient splicing reaction. On comparison with the X-ray crystal structure of the P4-5-6 domains of the Tetrahymena intron, three adenosines at positions 183, 184, and 186 were found to be identical to those significantly contributing to the formation of its tertiary structure. However, our results show that an adenosine at 187 is involved in the formation of a Watson-Crick base pair with U135, although it forms a Hoogsteen base pair in the crystal structure.

Multiple tertiary interactions involving domain II of group II self-splicing introns

The ribozyme core of group II introns is organized into six domains of secondary structure. Of these, domain II was long thought to be relatively unimportant for group II self-splicing. However, we now demonstrate the existence, in both major subdivisions of the group II family, of essential tertiary interactions involving domain II. θ-θ′ is a novel tertiary interaction between the terminal loop of the IC1 stem of domain I and the basal stem of domain II. The θ-θ′ interaction appears to stabilize the group II ribozyme core: it is essential for efficient self-splicing at elevated temperatures but, as shown by the use of a bimolecular reaction system, molecules with a defective θ-θ′ contact are not affected in catalysis. An interaction, η-η′, between domains II and VI of subgroup IIB introns was recently reported to mediate a conformational rearrangement between the two steps of the self-splicing reaction. We now show that domains II and VI of subgroup IIA introns also contact each other, although in a somewhat different way. Reinforcement of the η-η′ interaction of a subgroup IIA intron prevents the use of a specific 2′-hydroxyl group in domain VI to initiate splicing by transesterification at the 5′ splice site; the 5′ intron-exon junction is hydrolyzed instead. Since disruption of η-η′ has exactly opposite effects, and promotes reversal of the first transesterification step, it is concluded that formation of η-η′ mediates a conformational change in subgroup IIA introns as well. Just like the η-η′ interaction of subgroup IIB introns, the η-η′ interaction of subgroup IIA introns (and the θ-θ′ interaction) involves terminal loops of the GNRA family and their RNA receptors. Therefore, these motifs are used by nature not only to stabilize three-dimensional RNA architectures, but also in situations that require dynamic interactions.

Mechanistic investigations of a ribozyme derived from the Tetrahymena group I intron: insights into catalysis and the second step of self-splicing.

Self-splicing of Tetrahymena pre-rRNA proceeds in two consecutive phosphoryl transesterification steps. One major difference between these steps is that in the first an exogenous guanosine (G) binds to the active site, while in the second the 3'-terminal G414 residue of the intron binds. The first step has been extensively characterized in studies of the L-21ScaI ribozyme, which uses exogenous G as a nucleophile. In this study, mechanistic features involved in the second step are investigated by using the L-21G414 ribozyme. The L-21G414 reaction has been studied in both directions, with G414 acting as a leaving group in the second step and a nucleophile in its reverse. The rate constant of chemical step is the same with exogenous G bound to the L-21ScaI ribozyme and with the intramolecular guanosine residue of the L-21G414 ribozyme. The result supports the previously proposed single G-binding site model and further suggests that the orientation of the bound G and the overall active site structure is the same in both steps of the splicing reaction. An evolutionary rationale for the use of exogenous G in the first step is also presented. The results suggest that the L-21G414 ribozyme exists predominantly with the 3'-terminal G414 docked into the G-binding site. This docking is destabilized by approximately 100-fold when G414 is attached to an electron-withdrawing pA group. The internal equilibrium with K(int) = 0.7 for the ribozyme reaction indicates that bound substrate and product are thermodynamically matched and is consistent with a degree of symmetry within the active site. These observations are consistent with the presence of a second Mg ion in the active site. Finally, the slow dissociation of a 5' exon analog relative to a ligated exon analog from the L-21G414 ribozyme suggests a kinetic mechanism for ensuring efficient ligation of exons and raises new questions about the overall self-splicing reaction.

Conformational switches involved in orchestrating the successive steps of group I RNA splicing.

Group I introns possess a conserved guanosine residue at their 3' end, termed omegaG, that, in the case of the Tetrahymena pre-rRNA, is a major determinant of the second step of splicing. We examined the role of omegaG in self-splicing of the 249-residue group I intron of the Anabaena PCC7120 tRNAleu precursor. Contrary to observations with the Tetrahymena pre-rRNA intron, a mutation that places an adenosine residue at the omega position did not have a severe effect on the second step of splicing; neither 3' splice-site selection nor the rate of the second step was altered. The first step of splicing, however, was now readily reversed. This unexpected effect also resulted from a mutation that altered the nucleoside specificity of the intronic guanosine-binding site. The theme common to these mutations is that reversal of the first step of splicing results when there is not a strong interaction between the guanosine-binding site and the omega residue. This suggests that a major role of omegaG is to compete with the exogenous guanosine molecule added to the intron in the first step of splicing for the single guanosine-binding site of the intron. From these data, we are able to extend the mechanism for the self-splicing reaction of this intron by proposing two distinct conformational changes between the first and second steps of the splicing. The first of these is the exchange of the exogenous nucleoside for the omega nucleoside. This is the equilibrium that we can perturb by mutations at either the omega position or the guanosine-binding site. An additional conformational change then fully activates the intron for the second step of splicing.

Mechanisms of Intron Mobility

Mechanisms of Intron Mobility

Intron-containing T4 bacteriophage gene sunY encodes an anaerobic ribonucleotide reductase.

The function of the SunY protein, encoded by an intron-containing gene of bacteriophage T4, has remained hitherto unknown in contrast to the extensively studied self-splicing reaction of the SunY intron. Here we show that anaerobic T4 infections of Escherichia coli induce a ribonucleoside triphosphate reductase activity that is 10-30-fold higher than the bacterial host level of the corresponding enzyme. Inactivation of the T4 sunY gene (in this communication renamed nrdD) significantly decreased both the induced activity and the anaerobic production of phage, confirming the role of the T4 NrdD (SunY) protein as a phage-specific anaerobic ribonucleotide reductase. With the identification of the T4 nrdD (sunY) gene product as an anaerobic ribonucleotide reductase, all known bacteriophage introns are found to share the common and as yet unexplained property of residing within genes of DNA metabolism.

Evidence that the guanosine substrate of the Tetrahymena ribozyme is bound in the anti conformation and that N7 contributes to binding.

The group I self-splicing introns are RNA enzymes that catalyze phosphodiester-exchange reactions. These ribozymes have a highly specific binding site for guanosine, a substrate for the first self-splicing reaction (Bass & Cech, 1984). The binding site for guanosine has been localized to a specific region of the ribozyme (Michel et al., 1989), but the conformation of the bound guanosine substrate remains unknown. Most analogs of guanosine with substituents at C8 have a preference for the syn conformation; however, some C8-substituted analogs have the potential to form a hydrogen bond between the C8 substituent and the 5'-hydroxyl that would stabilize the anti conformation; we have found that analogs with the potential to form such a hydrogen bond are more active substrates than those that cannot form such a hydrogen bond. These observations led us to test 8-5'-O-cycloguanosine, which is locked in the anti conformation, and 8-(alpha-hydroxyisopropyl)guanosine, which is locked in the syn conformation; the former is active as a substrate, while the latter is inactive. These results strongly suggest that guanosine is bound to the ribozyme in the anti conformation and provide an additional constraint on structural models of this RNA enzyme. We have also examined a series of N7-substituted guanosine analogs; this position had previously been assumed to be unimportant for substrate binding since 7-methylguanosine is an excellent substrate. However, we have found that 7-deazaguanosine and 7-methyl-7-deazaguanosine are less active substrates than guanosine. We discuss several models for the role of N7 in guanosine binding.

Monitoring of the Cooperative Unfolding of the sunY Group I Intron of Bacteriophage T4 : The Active Form of the sunY Ribozyme is Stabilized by Multiple Interactions with 3′ Terminal Intron Components

We have studied the mechanism by which the 3′ terminal domain of the sunY intron of bacteriophage T4 activates the group I ribozyme core of this intron, from which it is separated by some 800 nucleotides. As shown by monitoring either UV absorbance or sell splicing reaction kinetics as a function of temperature, intron transcripts undergo highly cooperative unfolding/inactivation upon heating: the two methods yield similar estimates of the thermodynamic parameters associated with this process. Such cooperativity makes it possible in turn to assess the energetic contribution of specific interactions to the overall structure, by comparing the sensitivity to heat inactivation of molecules carrying various nucleotide substitutions. By combining this approach with chemical modification, we have probed several proven or putative interactions between the core and 3′ terminal domain of the intron and conclude that the role of the 3′ terminal domain is to stabilize the active form of the ribozyme. Interestingly, the P9.0 interaction, which brings 3′ terminal nucleotides next to the core site that hinds the guanosine cofactor of the self-splicing reaction, is now shown to be composed in fact of two distinct pairings. An isolated base-pair (P9.0a), involving a residue located only six nucleotides upstream of the 3′ splice site, participates in the stabilization of the ribozyme and appears to persist during the second stage of sell splicing (exon ligation). In contrast, formation of the previously demonstrated P9.0b pairing, which involves the two penultimate intron nucleotides, contributes no additional stability and results in no detectable rearrangement of the core structure. Implications for the concept of a static ribozyme are discussed in the light of a slightly revised three-dimensional model of the sunY intron.

Analysis of the Chloroplast Large Subunit Ribosomal RNA Gene from 17 Chlamydomonas Taxa : Three Internal Transcribed Spacers and 12 Group I Intron Insertion Sites

Previous reports on the chloroplast large subunit rRNA genes of the two distantly related green algae Chlamydomonas eugametos and Chlamydomonas reinhardtii indicate differences in the distribution of group I introns and suggest a different arrangement of internal transcribed spacers. To provide insights into the origin of these two types of intervening sequences, we have undertaken the sequencing of the chloroplast rrnL genes of 15 additional Chlamydomonas taxa and have characterized the mature large subunit rRNA species they encode in addition to those specified by the C. reinhardtii rrnL. These analyses disclosed the presence of three internal transcribed spacers sharing the same positions in all of the 17 taxa as well as the presence of a total of 39 group I introns representing 12 insertion sites. Of these insertion sites, only one has been identified in non- Chlamydomonas taxa. The distribution of Chlamydomonas introns is highly variable and, in many respects, is not consistent with the phylogeny deduced from chloroplast rRNA sequence comparisons. This phylogeny features two main lineages of Chlamydomonas taxa forming sister groups. Because earlier branching organisms in the green algal/land plant lineage display no chloroplast rDNA introns, it appears that all of the intron insertion positions in Chlamydomonas are of recent origins, with some of the positions having arisen subsequent to the divergence of the two main Chlamydomonas lineages. Remarkably, the rRNA regions corresponding to most of the group I intron insertion positions in rRNA genes have been assigned functional roles suggesting that they lie in exposed regions of the ribosome. On the basis of this striking correlation between exposed rRNA regions and intron insertion sites, we speculate that the reversal of the self-splicing reaction has played a major role in the creation of the multiple intron insertion positions found in rRNA genes as well as in the proliferation of group I introns elsewhere in the Chlamydomonas chloroplast genome.

Self-splicing of a Podospora anserina Group IIA Intron in vitro: Effects of 3′-Terminal Intron Alterations on Cleavage at the 5′ and 3′ Splice Site

A shortened derivative of the group IIA intron from the mitochondrial cytochrome-c-oxidase subunit I gene (COI II) of the ascomycete Podospora anserina can undergo self-splicing in vitro. When compared to self-splicing group IIB introns from yeast mitochondria (aI5c, bII) the autocatalytic reaction shows a lower efficiency and 5′ cleavage takes place predominantly by hydrolysis. In order to test the influence on reaction efficiency and mode of 5′ cleavage of the long peripheral structure of domain VI (dVI) we generated mutant Podospora introns that have different structural forms of shortened dVI. Our results show that: (1) in general the size and structure of dVI distal from the branch site is essential for 5′ transesterification and influences the efficiency of the second splicing step; (2) 5′ transesterification as well as the complete self-splicing reaction is more efficient when the structure of dVI is adapted to that of yeast group IIB introns. Moreover, our data indicate that the postulated γ-γ′ tertiary interaction is also functional for group IIA introns. A weakening or disruption of this interaction in the Podospora intron leads to a greatly reduced cleavage at the 3′ splice site and to a selection of cryptic sites downstream in the 3′ exon that almost exclusively restore the strong wild-type γ-γ′ pairing. The so-called "guide" interaction seems to support the selection of 3′ cleavage sites but is of secondary importance in relation to the γ-γ′ interaction.

Modeling Study on the Cleavage Step of the Self-Splicing Reaction in Group I Introns

A three-dimensional model of the Tetrahymena thermophila group I intron is used to further explore the catalytic mechanism of the transphosphorylation reaction of the cleavage step. Based on the coordinates of the catalytic core model proposed by Michel and Westhof (Michel, F., Westhof, E. J. Mol. Biol. 216, 585-610 (1990)), we first converted their ligation step model into a model of the cleavage step by the substitution of several bases and the removal of helix P9. Next, an attempt to place a trigonal bipyramidal transition state model in the active site revealed that this modified model for the cleavage step could not accommodate the transition state due to insufficient space. A lowering of P1 helix relative to surrounding helices provided the additional space required. Simultaneously, it provided a better starting geometry to model the molecular contacts proposed by Pyle et al. (Pyle, A. M., Murphy, F. L., Cech, T. R. Nature 358, 123-128. (1992)), based on mutational studies involving the J8/7 segment. Two hydrated Mg2+ complexes were placed in the active site of the ribozyme model, using the crystal structure of the functionally similar Klenow fragment (Beese, L.S., Steitz, T.A. EMBO J. 10, 25-33 (1991)) as a guide. The presence of two metal ions in the active site of the intron differs from previous models, which incorporate one metal ion in the catalytic site to fulfill the postulated roles of Mg2+ in catalysis. The reaction profile is simulated based on a trigonal bipyramidal transition state, and the role of the hydrated Mg2+ complexes in catalysis is further explored using molecular orbital calculations.

Characterization of the rRNA-encoding genes and transcripts, and a group-I self-splicing intron in Pneumocystis carinii

Although Pneumocystis carinii is the most common opportunistic pathogen infecting individuals with AIDS, very little is known of the basic biology of the organism. We have examined the ribosomal RNA (rRNA) and the DNA encoding it (rDNA) in P. carinii in an attempt to clarify its taxonomic position and to begin to study its genetic processes. Electrophoretic analysis showed that the sizes of the P. carinii rRNAs are quite similar to the sizes of the corresponding rRNAs from Saccharomyces cerevisiae. Direct sequence analysis of approx. 60% of the 18S small subunit-rRNA (Ss-rRNA) confirmed that its sequence is similar to that of yeast-like fungi and that a putative group-I intron previously observed in the 18S rDNA is, in fact, excised from the mature rRNA. PCR analysis of the intron in P. carinii genomic DNA showed that each of the multiple rDNA genes bears the group-I intron and in vitro transcripts of the intron autocatalytically excise from the rRNA primary transcript in the presence of GTP. Finally, analogues of GTP inhibit the self-splicing reaction, indicating that the guano sine-binding site of the intron closely resembles that of other well-characterized group-I introns. Since no group-I introns have been found in higher eukaryotes, this self-splicing process represents a viable target for chemotherapy of P. carinii pneumonia (PCP).