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T R Cech

Publications and source records attributed to T R Cech.

At least 145 records · Page 8Linked to original sources
PubMed1991

Ribozyme recognition of RNA by tertiary interactions with specific ribose 2'-OH groups.

Shortened forms of the group I intron from Tetrahymena catalyse sequence-specific cleavage of exogenous oligonucleotide substrates. The association between RNA enzyme (ribozyme) and substrate is mediated by pairing between an internal guide sequence on the ribozyme and a complementary sequence on the substrate. RNA substrates and cleavage products associate with a binding energy greater than that of base-pairing by approximately 4 kcal-mol-1 (at 42 degrees C), whereas DNA associates with an energy around that expected for base-pairing. It has been proposed that the difference in binding affinity is due to specific 2'-OH groups on an RNA substrate forming stabilizing tertiary interactions with the core of the ribozyme, or that the RNA.RNA helix formed upon association of an RNA substrate and the ribozyme might be more stable than an RNA.DNA helix of the same sequence. To differentiate between these two models, chimaeric oligonucleotides containing deoxynucleotide residues at successive positions along the chain were synthesized, and their equilibrium binding constants for association with the ribozyme were measured directly by a new gel electrophoresis technique. We report here that most of the extra binding energy can be accounted for by discrete RNA-ribozyme interactions, the 2'-OH group on the sugar residue three nucleotides from the cleavage site contributing the most interaction energy. Thus, in addition to the well documented binding of RNA to RNA by base-pairing, 2'-OH groups within a duplex can also mediate association between RNA molecules.

Animals↗
PubMed1991

Alternative secondary structures in the 5' exon affect both forward and reverse self-splicing of the Tetrahymena intervening sequence RNA.

The natural splice junction of the Tetrahymena large ribosomal RNA is flanked by hairpins that are phylogenetically conserved. The stem immediately preceding the splice junction involves nucleotides that also base pair with the internal guide sequence of the intervening sequence during splicing. Thus, precursors which contain wild-type exons can form two alternative helices. We have constructed a series of RNAs where the stem-loop in the 5' exon is more or less stable than in the wild-type precursor, and tested them in both forward and reverse self-splicing reactions. The presence of a stable hairpin in ligated exon substrates interferes with the ability of the intervening sequence to integrate at the splice junction. Similarly, the presence of the wild-type hairpin in the 5' exon reduces the rate of splicing 20-fold in short precursors. The data are consistent with a competition between unproductive formation of a hairpin in the 5' exon and productive pairing of the 5' exon with the internal guide sequence. The reduction of splicing by a hairpin that is a normal feature of rRNA structure is surprising; we propose that this attenuation is relieved in the natural splicing environment.

Animals↗
PubMed1991

Visualizing the higher order folding of a catalytic RNA molecule.

The higher order folding process of the catalytic RNA derived from the self-splicing intron of Tetrahymena thermophila was monitored with the use of Fe(II)-EDTA-induced free radical chemistry. The overall tertiary structure of the RNA molecule forms cooperatively with the uptake of at least three magnesium ions. Local folding transitions display different metal ion dependencies, suggesting that the RNA tertiary structure assembles through a specific folding intermediate before the catalytic core is formed. Enzymatic activity, assayed with an RNA substrate that is complementary to the catalytic RNA active site, coincides with the cooperative structural transition. The higher order RNA foldings produced by Mg(II), Ca(II), and Sr(II) are similar; however, only the Mg(II)-stabilized RNA is catalytically active. Thus, these results directly demonstrate that divalent metal ions participate in general folding of the ribozyme tertiary structure, and further indicate a more specific involvement of Mg(II) in catalysis.

Animals↗
PubMed1991

Binding of the CBP2 protein to a yeast mitochondrial group I intron requires the catalytic core of the RNA.

The yeast CBP2 gene product is required for the splicing of the terminal intron (bI5) of the mitochondrial cytochrome b pre-mRNA in vivo. In vitro, bI5 RNA self-splices efficiently only at high MgCl2 concentrations (50 mM); at 5 mM MgCl2, efficient splicing requires purified CBP2 protein. To determine the sequences within bI5 recognized by the protein, we have constructed deletion and substitution mutants of the RNA. Their binding to CBP2 was assessed by their ability to inhibit protein-dependent splicing of the wild-type bI5 RNA. Several regions, including the large L1 and L8 loops, can be deleted without affecting binding. They can therefore be eliminated from consideration as critical recognition elements. In contrast, other changes prevent the RNA from binding CBP2 and also impair self-splicing. Thus, either the catalytic core contacts the protein directly, or the integrity of the core is required for proper display of other RNA sequences that bind the protein. The results are consistent with a model in which the CBP2 protein facilitates splicing by binding to and stabilizing the active structure of the RNA. However, a more specific model is proposed in which the protein specifically enhances Mg2+ binding required for catalysis.

Base Sequence↗
PubMed1990

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 ribozyme derived from the intervening sequence (IVS) of the Tetrahymena preribosomal RNA catalyzes a site-specific endonuclease reaction: G2CCCUCUA5 + G in equilibrium with G2CCCUCU + GA5 (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 degrees C and neutral pH in the presence of 10 mM Mg2+. The value of kcat/Km = 9 x 10(7) M-1 min-1 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 RNA.RNA 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 kcat = 0.1 min-1 represents rate-limiting dissociation of the 5'-exon analogue, G2CCCUCU. The product GA5 dissociates first from the ribozyme because of this slow off-rate for G2CCCUCU. The similar binding of the product, G2CCCUCU, and the substrate, G2CCCUCUA5, to the 5'-exon binding site of the ribozyme, with Kd = 1-2 nM, shows that the pA5 portion of the substrate makes no net contribution to binding. Both the substrate and product bind approximately 10(4)-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 kc approximately equal to 350 min-1, a rate constant that is not reflected in the steady-state rate parameters with saturating G. The simplest interpretation is adopted, in which kc 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, kc(-G) = 0.7 min-1, is approximately 500-fold slower than that with saturating guanosine. The value of kcat/Km = 6 x 10(7) M-1 min-1 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.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 2. Kinetic description of the reaction of an RNA substrate that forms a mismatch at the active site.

The site-specific endonuclease reaction catalyzed by the ribozyme from the Tetrahymena pre-rRNA intervening sequence has been characterized with a substrate that forms a "matched" duplex with the 5' exon binding site of the ribozyme [G2CCCUCUA5 + G in equilibrium with G2CCCUCU + GA5 (G = guanosine); Herschlag, D., & Cech, T.R. (1990) Biochemistry (preceding paper in this issue)]. The rate-limiting step with saturating substrate is dissociation of the product G2CCCUCU. Here we show that the reaction of the substrate G2CCCGCUA5, which forms a "mismatched" duplex with the 5' exon binding site at position -3 from the cleavage site, has a value of kcat that is approximately 10(2)-fold greater than kcat for the matched substrate (50 degrees C, 10 mM MgCl2, pH 7). This is explained by the faster dissociation of the mismatched product, G2CCCGCU, than the matched product. With subsaturating oligonucleotide substrate and saturating G, the binding of the oligonucleotide substrate and the chemical step are each partially rate-limiting. The rate constant for the chemical step of the endonuclease reaction and the rate constant for the site-specific hydrolysis reaction, in which solvent replaces G, are each within approximately 2-fold with the matched and mismatched substrates, despite the approximately 10(3)-fold weaker binding of the mismatched substrate. This can be described as "uniform binding" of the base at position -3 in the ground state and transition state [Albery, W.J., & Knowles, J. R. (1976) Biochemistry 15, 5631-5640]. Thus, the matched substrate does not use its extra binding energy to preferentially stabilize the transition state.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Effect of monovalent cation-induced telomeric DNA structure on the binding of Oxytricha telomeric protein.

Oligonucleotides bearing 4 repeats of telomeric deoxyguanosine-rich sequence undergo a monovalent cation-induced transition to a folded conformation with G-G base pairs, modeled as a 'G-quartet' structure. We have now deduced the rates of folding and unfolding of d(TTTTGGGG)4, which has four repeats of the Oxytricha telomeric DNA sequence. The estimated average values of delta G for the folded form at 37 degrees C are -2.2 kcal/mol and -4.7 kcal/mol in 50 mM na+ and K+, respectively. The fully folded DNA is not recognized by the Oxytricha telomere-binding protein; the substrate for protein binding has properties consistent with its being partly or fully unfolded. In confirmation of this conclusion, prevention of DNA folding by methylation enables the protein to bind as rapidly in the presence of monovalent cations as in their absence. The slow unfolding (t1/2 = 4 hr and 18 hr at 37 degrees C in Na+ and K+, respectively) of the DNA suggests that such structures would be long-lived if they formed in vivo, unless they can be actively unfolded. The inability of the telomere-binding protein to bind the stable, folded form of the 4-repeat telomeric sequence is a problem that may be circumvented in vivo by avoiding four single-stranded repeats.

Base Composition↗
PubMed1990

An ultraviolet-inducible adenosine-adenosine cross-link reflects the catalytic structure of the Tetrahymena ribozyme.

When a shortened enzymatic version of the Tetrahymena self-splicing intervening sequence (IVS) RNA is placed under catalytic conditions and irradiated at 254 nm, a covalent cross-link forms with high efficiency. The position of the cross-link was mapped by using three independent methods: RNase H digestion, primer extension with reverse transcriptase, and partial hydrolysis of end-labeled RNA. The cross-link is chemically unusual in that it joins two adenosines, A57 and A95. Formation of this cross-link depends upon the identity and concentration of divalent cations present and upon heat-cool renaturation of the IVS in a manner that parallels conditions required for optimal catalytic activity. Furthermore, cross-linking requires the presence of sequences within the core structure, which is conserved among group I intervening sequences and necessary for catalytic activity. Together these correlations suggest that a common folded structure permits cross-linking and catalytic activity. The core can form this structure independent of the presence of P1 and elements at the 3' end of the IVS. The cross-linked RNA loses catalytic activity under destabilizing conditions, presumably due to disruption of the folded structure by the cross-link. One of the nucleotides participating in this cross-link is highly conserved (86%) within the secondary structure of group I intervening sequences. We conclude that A57 and A95 are precisely aligned in a catalytically active conformation of the RNA. A model is presented for the tertiary arrangement in the vicinity of the cross-link.

Adenosine↗
PubMed1990

DNA cleavage catalysed by the ribozyme from Tetrahymena.

An RNA enzyme derived from the self-splicing intervening sequence of Tetrahymena thermophila catalyses sequence-specific cleavage of an oligodeoxyribonucleotide substrate. Compared with RNA, the DNA substrate is bound very weakly and is cleaved very slowly, revealing the importance of the RNA 2'-hydroxyl group in both the binding and chemical steps. The finding that catalysis by RNA can extend to DNA substrates indicates new possibilities for the transposition of intervening sequences and for the design of DNA cleavage agents with novel sequence specificities.

Animals↗
PubMed1990

Iron(II)-ethylenediaminetetraacetic acid catalyzed cleavage of RNA and DNA oligonucleotides: similar reactivity toward single- and double-stranded forms.

Fe(II)-EDTA catalyzes the cleavage of nucleic acids with little or no base-sequence specificity. We have now studied the preference of this reagent in catalyzing the cleavage of single- versus double-stranded nucleic acid structures. Three RNA and two DNA molecules, each expected to contain both single- and double-stranded regions, were synthesized and their structures characterized by enzymatic digestion using secondary structure specific nucleases. Fe(II)-EDTA catalyzed nearly uniform strand scission along the entire length of each molecule; no correlation with secondary structure was observed. The homopolymer sequence dA30:dT30, embedded in a mixed-sequence context to promote exact register of the homopolymer tract, was cleaved to an extent similar to that of flanking sequences. The reactions were relatively insensitive to K+, Na+, and Mg2+ in the range 10-100 mM and were quenched by Tris-HCl buffer. We conclude that the Fe(II)-EDTA-catalyzed strand scission reaction does not discriminate between typical single- and double-stranded regions, which simplifies the interpretation of experiments in which the reaction is used to probe the tertiary structure of RNA molecules [Latham, J. A., & Cech, T. R. (1989) Science 245, 276-282].

Base Sequence↗
PubMed1990

Nobel lecture. Self-splicing and enzymatic activity of an intervening sequence RNA from Tetrahymena.

A living cell requires thousands of different chemical reactions to utilize energy, move, grow, respond to external stimuli and reproduce itself. While these reactions take place spontaneously, they rarely proceed at a rate fast enough for life. Enzymes, biological catalysts found in all cells, greatly accelerate the rates of these chemical reactions and impart on them extraordinary specificity. In 1926, James B. Summer crystallized the enzyme urease and found that it was a protein. Skeptics argued that the enzymatic activity might reside in a trace component of the preparation rather than in the protein (Haldane, 1930), and it took another decade for the generality of Summer's finding to be established. As more and more examples of protein enzymes were found, it began to appear that biological catalysis would be exclusively the realm of proteins. In 1981 and 1982, my research group and I found a case in which RNA, a form of genetic material, was able to cleave and rejoin its own nucleotide linkages. This self-splicing RNA provided the first example of a catalytic active site formed of ribonucleic acid. This lecture gives a personal view of the events that led to our realization of RNA self-splicing and the catalytic potential of RNA. It provides yet another illustration of the circuitous path by which scientific inquiry often proceeds. The decision to expand so many words describing the early experiments means that much of our current knowledge about the system will not be mentioned. For a more comprehensive view of the mechanism and structure of the Tetrahymena self-splicing RNA and RNA catalysis in general, the reader is directed to a number of recent reviews (Cech & Bass, 1986: Cech, 1987, 1988a, 1990; Burke, 1988; Altman, 1989). Possible medical and pharmaceutical implications of RNA catalysis have also been described recently (Cech, 1988b).

Animals↗
PubMed1990

Direct measurement of oligonucleotide substrate binding to wild-type and mutant ribozymes from Tetrahymena.

Like protein enzymes, RNA enzymes (ribozymes) provide specific binding sites for their substrates. We now show that equilibrium dissociation constants for complexes between the Tetrahymena ribozyme and its RNA substrates and products can be directly measured by electrophoresis in polyacrylamide gels containing divalent cations. Binding is 10(3)- to 10(4)-fold tighter (4-5 kcal/mol at 42 degrees C) than expected from base-pairing interactions alone, implying that tertiary interactions also contribute to energetic stabilization. Binding decreases with single base changes in the substrate, substitution of deoxyribose sugars, and lower Mg2+ concentration. Ca2+, which enables the ribozyme to fold but is unable to mediate efficient RNA cleavage, promotes weaker substrate binding than Mg2+. This indicates that Mg2+ has special roles in both substrate binding and catalysis. Mutagenesis of a region near the internal guide sequence disrupts substrate binding, whereas binding is not significantly affected by a mutation of the guanosine-binding site. This approach should be generally useful for analysis of ribozyme variants independent of their catalytic activities.

Animals↗
PubMed1990

Two versions of the gene encoding the 41-kilodalton subunit of the telomere binding protein of Oxytricha nova.

Macronuclear chromosomes of the ciliated protozoan Oxytricha nova terminate with a single-stranded (T4G4)2 overhang. The (T4G4)2 telomeric overhang is tenaciously bound by a protein heterodimer. We have cloned and sequenced the gene encoding the 41-kDa subunit of this telomere binding protein. The predicted amino acid sequence comprises two distinct regions, a carboxyl-terminal two-thirds that is 23% lysine and bears similarity to histone H1 and an amino-terminal one-third containing a hydrophobic stretch of about 15 amino acids. Two macronuclear versions of the gene differ in nucleotide sequence at several positions, but the derived polypeptides differ only at a single position, Ser-110 or Ala-110. Both versions harbor a small intron. The existence of this intron demonstrates that, despite the elimination of 95% of the micronuclear genome from the developing macronucleus, at least some noncoding DNA is retained during macronuclear development of hypotrichous ciliates.

Amino Acid Sequence↗
PubMed1989

Monovalent cation-induced structure of telomeric DNA: the G-quartet model.

We have investigated the structures formed by oligonucleotides composed of two or four repeats of the telomeric sequences from Oxytricha and Tetrahymena. The Oxytricha four-repeat molecule (d(T4G4)4 = Oxy-4) forms structures with increased electrophoretic mobility in nondenaturing gels containing Na+, K+, or Cs+, but not in gels containing Li+ or no added salt. Formation of the folded structure results in protection of a set of dG's from methylation by dimethyl sulfate. Efficient UV-induced cross-links are observed in Oxy-4 and the related sequence from Tetrahymena (d(T2G4)4 = Tet-4), and join thymidine residues in different repeats. Models proposed to account for these data involve G-quartets, hydrogen-bonded structures formed from four guanosine residues in a square-planar array. We propose that the G-quartet structure must be dealt with in vivo by the telomere replication machinery.

Animals↗
PubMed1989

Assembly and self-association of oxytricha telomeric nucleoprotein complexes.

Two types of specific telomeric protein-DNA complex are reconstituted upon incubation of purified Oxytricha telomere protein with (T4G4)4, an oligodeoxynucleotide of telomeric sequence. The complexes differ in electrophoretic mobility, in protein-DNA contacts, and in the rate of DNA exchange. The patterns of protein-DNA interaction determined by modification interference suggest a model in which the protein can bind either to the two T4G4 repeats at the 3' end or to two internal repeats; in the latter case, it can make a different set of contacts with the terminal repeat to form the more stable complex. Native telomeric chromatin isolated from Oxytricha contains both types of complexes. The reconstituted monomeric complexes associate to give a high molecular weight form that has an altered chemical footprint. Such interactions may mediate the association of chromosomal telomeres in vivo.

Animals↗