Role of conserved sequence elements 9L and 2 in self-splicing of the Tetrahymena ribosomal RNA precursor

Burke, John M.; Irvine, Kenneth D.; Kaneko, Kotaro; Kerker, Barbara J.; Oettgen, A.Barbara; Tierney, William M.; Williamson, Cynthia L.; Zaug, Arthur J.; Cech, Thomas R.

doi:10.1016/0092-8674(86)90380-6

Cited by 79 publications

(48 citation statements)

References 44 publications

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“…Approximately 300 site-specific mutations of the Tetrahymena intervening sequence have been tested for their effect on RNA splicing or on cleavage at the 5'-splice site, and additional mutations have been studied in other group I intervening sequences [e.g., Waring et al (1989, Burke et al, (1986, Been et al (1987), Belfort et al (1987), Flor et al…”

Section: Conclusion and Future Prospectsmentioning

confidence: 99%

Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 1. Kinetic description of the reaction of an RNA substrate complementary to the active site

Herschlag

Cech²

1990

Biochemistry

Self Cite

317

481

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A ribozyme derived from the intervening sequence (IVS) of the Tetrahymenit preribosomal RNA catalyzes a site-specific endonuclease reaction: G2CCCUCUA5 + G + G2CCCUCU + GAS (G = guanosine). This reaction is analogous to the first step in self-splicing of the pre-rRNA, with the product G2CCCUCU analogous to the 5'-exon. The following mechanistic conclusions have been derived from pre-steady-state and steady-state kinetic measurements at 50 " C and neutral pH in the presence of 10 mM Mg2+. The value of k,,/Km = 9 X lo7 M-' min-' for the oligonucleotide substrate with saturating G represents rate-limiting binding. This rate constant for binding is of the order expected for formation of a RNAaRNA duplex between oligonucleotides. (Phylogenetic and mutational analyses have shown that this substrate is recognized by base pairing to a complementary sequence within the IVS.) The value of k, , = 0.1 min-' represents rate-limiting dissociation of the 5'-exon analogue, G2CCCUCU. The product GAS dissociates first from the ribozyme because of this slow off-rate for G2CCCUCU. The similar binding of the product, G2CCCUCU, and the substrate, G2CCCUCUAS, to the 5'-exon binding site of the ribozyme, with Kd = 1-2 nM, shows that the pAs portion of the substrate makes no net contribution to binding. Both the substrate and product bind -104-fold (6 kcal/mol) stronger than expected from base pairing with the 5'-exon binding site. Thus, tertiary interactions are involved in binding. Binding of G2CCCUCU and binding of G are independent. These and other data suggest that binding of the oligonucleotide substrate, G2CCCUCUA5, and binding of G are essentially random and independent. The rate constant for reaction of the ternary complex is calculated to be k, 350 m i d , a rate constant that is not reflected in the steady-state rate parameters with saturating G. The simplest interpretation is adopted, in which k, represents the rate of the chemical step. A site-specific endonuclease reaction catalyzed by the Tetrahymena ribozyme in the absence of G was observed; the rate of the chemical step with solvent replacing guanosine, k,(-G) = 0.7 m i d , is -500-fold slower than that with saturating guanosine. The value of k,,/Km = 6 X lo7 M-' m i d for this hydrolysis reaction is only slightly smaller than that with saturating guanosine, because the binding of the oligonucleotide substrate is predominantly rate-limiting in both cases. This ribozyme, which approaches the limiting values of k,,/Km for protein enzymes, can be considered to have achieved "catalytic perfection"

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Section: Conclusion and Future Prospectsmentioning

confidence: 99%

Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 1. Kinetic description of the reaction of an RNA substrate complementary to the active site

Herschlag

Cech²

1990

Biochemistry

Self Cite

317

481

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“…3). The restoration of activity with higher concentrations of magnesium cation has been observed in a number of mutants of the Tetrahymena intron (Burke et al 1986;Flor et al 1989;Joyce et al 1989) and has been attributed to the stabilization of a destabilized structure. Also, an RNA enzyme derived from the Tetrahymena intron retains its ability to cleave an RNA substrate even after the As7-A9s cross-link has been introduced (W. Downs and T. Cech, unpubl.).…”

Section: Asz and Ags Contribute Structural Stabilitymentioning

confidence: 99%

“…Once again a U. G pair is required for cleavage, but in this case it is composed of U_s and G22. To achieve this, the 5' exon strand of P 1 must slide relative to the IGS, as has been proposed previously Zaug and Cech 1986). In this model the IGS remains fixed in position relative to the nucleophile, and only the U that base-pairs with Gx2 changes (Fig.…”

Section: Correlations Between 5'ss + 14 and 5'ss-4 Cleavages And Cyclmentioning

confidence: 99%

“…Other mutations decrease the rate of reaction without activating cleavage at positions -4 and + 14, including base pair changes and mismatches in P7 (Burke et al 1986) and in P4 (Flor et al 1989), and mutations near the 3' splice site that reduce 5' splice-site activity (Price and Cech 1988). This supports our interpretation that As7 and Ags participate specifically in proper positioning of the 5' splice site, as do U_ ~ "G22 (A. Pyle, T. Chapman, T. Cech, D. Turner, and S. Moran, in prep.…”

Section: Asz and Ags Interact To Promote Accurate Splicingmentioning

confidence: 99%

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A tertiary interaction in the Tetrahymena intron contributes to selection of the 5' splice site.

Downs¹,

Cech²

1994

Genes Dev.

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The utilization of cryptic splice sites has been observed in a number of RNA splicing reactions. In the self-splicing group I intron of Tetrahymena thermophila, point mutations of either As7 or A9s promote cleavage at two sites other than the normal 5' splice site, suggesting that these nucleotides are involved in a common tertiary interaction. These results are unusual since As7 and A9s are neither at nor near the 5' splice site in the sequence or secondary structure. Cleavage at the alternative sites appears to occur by intron cyclization, a reaction with well-established structural and mechanistic similarities to the first step of RNA self-splicing. Alternative docking of Pl (the helix containing the 5' splice site paired to the internal guide sequence of the intron) into the catalytic core accounts for cleavage at the cryptic reaction sites. We propose that the AsT/A9s interaction, along with an element implicated previously (J1/2), provide structural connectivity from the reaction site in Pl to the catalytic core of the Tetrahymena intron. It seems likely that RNA splicing in general will require such tertiary interactions to position RNA helices.[Key Words: Accuracy; group I introns; photo-cross-linking; ribozyme; RNA splicing; RNA tertiary structure] Received March 11, 1994; revised version accepted April 12, 1994.In spliceosomal mRNA splicing and in self-splicing, RNA makes up part or all of the splicing machinery. In each case, the first chemical step is cleavage at the 5' splice site, a reaction that must occur with high accuracy. Our understanding of 5' splice site selection has focused on base-pairing interactions, and in each case RNA components of the splicing machinery have been found to provide binding sites or "guide sequences" that base-pair with sequence flanking the 5' splice site. In group I and group II self-splicing reactions, the introns themselves provide the base-pairing sequences (Davies et al. 1982;Michel and Dujon 1983;Michel et al. 1989b). In nuclear mRNA splicing the initial recognition is by the U1 small nuclear RNA (snRNA} (Lerner et al. 1980;Zhuang and Weiner 1986;Guthrie 1991), later to be replaced by base-pairing with U5 and U6 snRNAs (Kandels-Lewis and S6raphin 1993; Lesser and Guthrie 1993; Sontheimer and Steitz 1993). Whereas these base-pairing interactions are necessary to direct the first step of splicing, it seems unlikely that they alone could ensure accuracy; once the splice-site duplex is formed, some mechanism is needed to align it with respect to the attacking group, which is contained in a second RNA du- plex (P7 in group I introns, domain VI in group II introns, the U2 snRNA, branch site duplex in nuclear mRNA introns). We now identify a tertiary interaction in the Tetrahymena group I intron that helps to provide this next level of accuracy by holding the 5' splice-site duplex into the proper position for splicing.Self-splicing of the Tetrahymena pre-rRNA and cyclization of the excised intron occur in three steps, each involving the same chemical mechanism of tran...

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“…The protein-dependent reaction utilizes a guanosine nucleoside cofactor for 5' splice site cleavage and generates products identical to those detected under self-splicing conditions. Furthermore, a structural element in the RNA previously shown to be necessary for catalytic activity in self-splicing (5,39,45) is also essential for protein-dependent splicing.…”

mentioning

confidence: 99%

CBP2 protein promotes in vitro excision of a yeast mitochondrial group I intron.

Gampel¹,

Nishikimi²,

Tzagoloff³

1989

Mol. Cell. Biol.

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The terminal intron (b12) of the yeast mitochondrial cytochrome b gene is a group I intron capable of self-splicing in vitro at high concentrations of Mg2+. Excision of b12 in vivo, however, requires a protein encoded by the nuclear gene CBP2. The CBP2 protein has been partially purified from wild-type yeast mitochondria and shown to promote splicing at physiological concentrations of Mg2+. The self-splicing and protein-dependent splicing reactions utilized a guanosine nucleoside cofactor, the hallmark of group I intron self-splicing reactions. Furthermore, mutations that abolished the autocatalytic activity of b12 also blocked protein-dependent splicing. These results indicated that protein-dependent excision of b12 is an RNA-catalyzed process involving the same two-step transesterification mechanism responsible for self-splicing of group I introns. We propose that the CBP2 protein binds to the bI2 precursor, thereby stabilizing the catalytically active structure of the RNA. The same or a similar RNA structure is probably induced by high concentrations of Mg2e in the absence of protein. Binding of the CBP2 protein to the unspliced precursor was supported by the observation that the protein-dependent reaction was saturable by the wild-type precursor. Proteindependent splicing was competitively inhibited by excised bI2 and by a splicing-defective precursor with a mutation in the 5' exon near the splice site but not by a splicing-defective precursor with a mutation in the core structure. Binding of the CBP2 protein to the precursor RNA had an effect on the 5' splice site helix, as evidenced by suppression of the interaction of an exogenous dinucleotide with the internal guide sequence.A substantial number of genes in fungal mitochondrial DNA contain group I introns (35). Although some mitochondrial group I introns are able to self-splice (splice in the absence of protein) in vitro, there is compelling evidence that in vivo splicing requires the participation of protein factors. In Saccharomyces cerevisiae, at least 11 nuclear gene products have been reported to be required for splicing of mitochondrial transcripts (27,33,40). In addition, some group I introns in yeast mitochondrial genes contain open reading frames that code for proteins (maturases) involved in the excision of the cognate intervening sequences (IVSs) from the pre-mRNAs (2, 12, 29). Protein-facilitated splicing is not confined to yeast mitochondrial transcripts. Excision of several mitochondrial group I introns of Neurospora crassa depends on a protein encoded by the nuclear gene cytl8 (9). This protein has been partially purified and shown to promote splicing of the large rRNA precursor in vitro (1, 16).The terminal intron (bI2) of the yeast mitochondrial cytochrome b pre-mRNA is an example of a group I intron whose in vivo excision depends on a nuclear gene product (bI2 of the short cytochrome b gene is identical to b15 of the long gene). Mutations in a gene designated CBP2 have been shown to selectively block processing of b12 (23, 33). In vitro...

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Role of conserved sequence elements 9L and 2 in self-splicing of the Tetrahymena ribosomal RNA precursor

Cited by 79 publications

References 44 publications

Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 1. Kinetic description of the reaction of an RNA substrate complementary to the active site

Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 1. Kinetic description of the reaction of an RNA substrate complementary to the active site

A tertiary interaction in the Tetrahymena intron contributes to selection of the 5' splice site.

CBP2 protein promotes in vitro excision of a yeast mitochondrial group I intron.

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