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T R Cech

Publications and source records attributed to T R Cech.

At least 127 records · Page 7Linked to original sources
PubMed1993

Metal ion catalysis in the Tetrahymena ribozyme reaction.

All catalytic RNAs (ribozymes) require or are stimulated by divalent metal ions, but it has been difficult to separate the contribution of these metal ions to formation of the RNA tertiary structure from a more direct role in catalysis. The Tetrahymena ribozyme catalyses cleavage of exogenous RNA or DNA substrates with an absolute requirement for Mg2+ or Mn2+ (ref. 6). A DNA substrate, in which the bridging 3' oxygen atom at the cleavage site is replaced by sulphur, is cleaved by the ribozyme about 1,000 times more slowly than the corresponding unmodified DNA substrate when Mg2+ is present as the only divalent metal ion. But addition of Mn2+ or Zn2+ to the reaction relieves this negative effect, with the 3' S-P bond being cleaved nearly as fast as the 3' O-P bond. Considering that Mn2+ and Zn2+ coordinate sulphur more strongly than Mg2+ does, these results indicate that the metal ion contributes directly to catalysis by coordination to the 3' oxygen atom in the transition state, presumably stabilizing the developing negative charge on the leaving group. We conclude that the Tetrahymena ribozyme is a metalloenzyme, with mechanistic similarities to several protein enzymes.

Animals↗
PubMed1993

Oxytricha telomere-binding protein: separable DNA-binding and dimerization domains of the alpha-subunit.

A telomere-binding protein heterodimer of 56-kD (alpha) and 41-kD (beta) subunits binds to the single-stranded (T4G4)2 terminus of each Oxytricha nova macronuclear DNA molecule. The alpha-subunit by itself binds to telomeric DNA. The beta-subunit alone does not bind to DNA specifically but interacts with the alpha-subunit to form a very stable ternary complex. We show that the formation of alpha-beta-DNA ternary complex is extremely cooperative. Furthermore, the binary complex (alpha-DNA) has a dissociation half-life of much less than 1 min; addition of the beta-subunit increases the half-life to approximately 100 hrs. Libraries of plasmids with random deletions of the open reading frame for the alpha-subunit were introduced into Escherichia coli, and extracts were subsequently checked for both protein expression and DNA-binding activity with or without added beta-subunit. The alpha-subunit was found to contain two structurally separable domains with distinct functions. The amino-terminal two-thirds is necessary and sufficient for sequence-specific DNA binding. The carboxy-terminal one-third is responsible for alpha/beta-subunit interactions. When expressed separately in E. coli, purified, and mixed together, these two domains reconstitute the activity of the wild-type alpha-subunit (trans-complementation in vitro). The amino-terminal two-thirds of the beta-subunit is necessary and sufficient both for alpha/beta-subunit interactions and for ternary complex formation. We conclude that the alpha-subunit of the telomere-binding protein, like many transcription factors, has separable DNA-binding and protein-protein interaction domains.

Animals↗
PubMed1993

Nuclear proteins that bind the pre-mRNA 3' splice site sequence r(UUAG/G) and the human telomeric DNA sequence d(TTAGGG)n.

HeLa cell nuclear proteins that bind to single-stranded d(TTAGGG)n, the human telomeric DNA repeat, were identified and purified by a gel retardation assay. Immunological data and peptide sequencing experiments indicated that the purified proteins were identical or closely related to the heterogeneous nuclear ribonucleoproteins (hnRNPs) A1, A2-B1, D, and E and to nucleolin. These proteins bound to RNA oligonucleotides having r(UUAGGG) repeats more tightly than to DNA of the same sequence. The binding was sequence specific, as point mutation of any of the first 4 bases [r(UUAG)] abolished it. The fraction containing D and E hnRNPs was shown to bind specifically to a synthetic oligoribonucleotide having the 3' splice site sequence of the human beta-globin intervening sequence 1, which includes the sequence UUAGG. Proteins in this fraction were further identified by two-dimensional gel electrophoresis as D01, D02, D1*, and E0; intriguingly, these members of the hnRNP D and E groups are nuclear proteins that are not stably associated with hnRNP complexes. These studies establish the binding specificities of these D and E hnRNPs. Furthermore, they suggest the possibility that these hnRNPs could perhaps bind to chromosome telomeres, in addition to having a role in pre-mRNA metabolism.

Amino Acid Sequence↗
PubMed1992

Mutations at the guanosine-binding site of the Tetrahymena ribozyme also affect site-specific hydrolysis.

Self-splicing group I introns use guanosine as a nucleophile to cleave the 5' splice site. The guanosine-binding site has been localized to the G264-C311 base pair of the Tetrahymena intron on the basis of analysis of mutations that change the specificity of the nucleophile from G (guanosine) to 2AP (2-aminopurine ribonucleoside) (F. Michel et al. (1989) Nature 342, 391-395). We studied the effect of these mutations (G-U, A-C and A-U replacing G264-C311) in the L-21 ScaI version of the Tetrahymena ribozyme. In this enzymatic system (kcat/Km)G monitors the cleavage step. This kinetic parameter decreased by at least 5 x 10(3) when the G264-C311 base pair was mutated to an A-U pair, while (kcat/Km)2AP increased at least 40-fold. This amounted to an overall switch in specificity of at least 2 x 10(5). The nucleophile specificity (G > 2AP for the G-C and G-U pairs, 2AP > G for the A-U and A-C pairs) was consistent with the proposed hydrogen bond between the nucleotide at position 264 and N1 of the nucleophile. Unexpectedly, the A-U and A-C mutants showed a decrease of an order of magnitude in the rate of ribozyme-catalyzed hydrolysis of RNA, in which H2O or OH- replaces G as the nucleophile, whereas the G-U mutant showed a decrease of only 2-fold. The low hydrolysis rates were not restored by raising the Mg2+ concentration or lowering the temperature. In addition, the mutant ribozymes exhibited a pattern of cleavage by Fe(II)-EDTA indistinguishable from that of the wild type, and the [Mg2+]1/2 for folding of the A-U mutant ribozyme was the same as that of the wild type. Therefore the guanosine-binding site mutations do not appear to have a major effect on RNA folding or stability. Because changing G264 affects the hydrolysis reaction without perturbing the global folding of the RNA, we conclude that the catalytic role of this conserved nucleotide is not limited to guanosine binding.

Animals↗
PubMed1992

RNA substrate binding site in the catalytic core of the Tetrahymena ribozyme.

In catalysis by group I introns, the helix (P1) containing the RNA cleavage site must be positioned next to the guanosine binding site. We have identified a conserved adenine in the catalytic core that contributes to the stability of this arrangement and propose that it accepts a hydrogen bond from a specific 2'-OH in P1. Such base-backbone tertiary interactions may be generally important to the organization of RNA tertiary structure.

Adenine↗
PubMed1992

Aminoacyl esterase activity of the Tetrahymena ribozyme.

Several classes of ribozymes (catalytic RNA's) catalyze reactions at phosphorus centers, but apparently no reaction at a carbon center has been demonstrated. The active site of the Tetrahymena ribozyme was engineered to bind an oligonucleotide derived from the 3' end of N-formyl-methionyl-tRNA(fMet). This ribozyme catalyzes the hydrolysis of the aminoacyl ester bond to a modest extent, 5 to 15 times greater than the uncatalyzed rate. Catalysis involves binding of the oligonucleotide to the internal guide sequence of the ribozyme and requires Mg2+ and sequence elements of the catalytic core. The ability of RNA to catalyze reactions with aminoacyl esters expands the catalytic versatility of RNA and suggests that the first aminoacyl tRNA synthetase could have been an RNA molecule.

Animals↗
PubMed1992

Tertiary structure around the guanosine-binding site of the Tetrahymena ribozyme.

A cleavage reagent directed to the active site of the Tetrahymena catalytic RNA was synthesized by derivatization of the guanosine substrate with a metal chelator. When complexed with iron(II), this reagent cleaved the RNA in five regions. Cleavage at adenosine 207, which is far from the guanosine-binding site in the primary and secondary structure, provides a constraint for the higher order folding of the RNA. This cleavage site constitutes physical evidence for a key feature of the Michel-Westhof model. Targeting a reactive entity to a specific site should be generally useful for determining proximity within folded RNA molecules or ribonucleoprotein complexes.

Animals↗
PubMed1991

Folding of group I introns from bacteriophage T4 involves internalization of the catalytic core.

Fe(II)-EDTA, a solvent-based cleavage reagent that distinguishes between the inside and outside surfaces of a folded RNA molecule, has revealed some of the higher-order folding of the group IB intron from Tetrahymena thermophila pre-rRNA. This reagent has now been used to analyze the bacteriophage T4 sunY and td introns, both of which are members of the group IA subclass. Significant portions of the phylogenetically conserved secondary structure are protected from Fe(II)-EDTA cleavage. However, the P4 secondary structure element, which is substantially protected in the Tetrahymena intron, is available for cleavage in the two T4 introns. We conclude that a family of catalytic RNAs (ribozymes) that possess similar secondary structures and have similar activities fold into similar but nonidentical tertiary structures that nevertheless serve to internalize portions of the catalytic center. Furthermore, comparison of cleavage patterns of the sunY and td intron RNAs indicates that conserved nucleotides outside as well as within the catalytic core participate in the tertiary structure.

Animals↗
PubMed1991

Mutations in a nonconserved sequence of the Tetrahymena ribozyme increase activity and specificity.

The RNA substrate-binding site of the Tetrahymena ribozyme is connected to the catalytic core by the joining region J1/2. Although J1/2 is not conserved among group I introns, small insertions or deletions in this sequence have dramatic effects, enhancing the turnover number and sequence specificity of ribozyme-catalyzed RNA cleavage. Measurements of rate constants for individual steps in the reaction have revealed the basis of these improvements. Ironically, the higher turnover and specificity both result from decreased affinity for RNA, rather than better cleavage. These results provide evidence that the nonconserved J1/2 sequence positions the RNA substrate to optimize tertiary interactions and ensure cleavage at the position corresponding to the 5' splice site. The wild-type RNA is well adapted to its biological function, and its limitations in multiple turnover can be corrected by mutation.

Animals↗
PubMed1991

Cloning and expression of genes for the Oxytricha telomere-binding protein: specific subunit interactions in the telomeric complex.

Telomeres of Oxytricha nova macronuclear chromosomes consist of a repeated T4G4 sequence, single-stranded at the 3' terminus, bound by a heterodimeric protein. The cloning of genes for the two polypeptides and their separate expression in E. coli have enabled evaluation of their individual contributions to DNA binding. The 56 kd alpha subunit binds single-stranded DNA by itself, one polypeptide per T4G4 block; multiple subunits can coat a (T4G4)n multimer. The derived amino acid sequence of alpha does not reveal any known DNA-binding motif, so it appears to represent a novel type of DNA-binding protein. The previously cloned 41 kd beta subunit does not by itself protect DNA from methylation, but is required along with alpha to recreate the pattern of methylation protection indicative of telomeres in vivo. The unusual ability of the protein to engage in two different interactions with the same telomeric DNA sequence might provide the versatility necessary for diverse telomere functions.

Amino Acid Sequence↗
PubMed1991

Molecular cloning of telomere-binding protein genes from Stylonychia mytilis.

A telomere-binding protein heterodimer of 56 kDa (alpha) and 41 kDa (beta) subunits binds specifically to Oxytricha nova telomeres. Genes encoding both subunits have been cloned previously. Here we report molecular cloning and sequence analysis of the homologous genes in Stylonychia mytilis. The derived amino acid sequences were 79% identical for the alpha subunits and 77% identical for the beta subunits. Three repeats of a Leu/Ile heptad were found in each subunit, which might be involved in heterodimer formation. A 360 amino acid region of the Stylonychia mytilis alpha subunit was found to share weak sequence similarity with human vimentin, suggesting the possibility of a relationship between telomeres and intermediate filaments.

Amino Acid Sequence↗
PubMed1991

A self-splicing group I intron in the nuclear pre-rRNA of the green alga, Ankistrodesmus stipitatus.

The nuclear small subunit ribosomal RNA gene of the unicellular green alga Ankistrodesmus stipitatus contains a group I intron, the first of its kind to be found in the nucleus of a member of the plant kingdom. The intron RNA closely resembles the group I intron found in the large subunit rRNA precursor of Tetrahymena thermophila, differing by only eight nucleotides of 48 in the catalytic core and having the same peripheral secondary structure elements. The Ankistrodesmus RNA self-splices in vitro, yielding the typical group I intron splicing intermediates and products. Unlike the Tetrahymena intron, however, splicing is accelerated by high concentrations of monovalent cations and is rate-limited by the exon ligation step. This system provides an opportunity to understand how limited changes in intron sequence and structure alter the properties of an RNA catalytic center.

Animals↗
PubMed1991

A hammerhead ribozyme allows synthesis of a new form of the Tetrahymena ribozyme homogeneous in length with a 3' end blocked for transesterification.

The L-21 Scal form of the Tetrahymena ribozyme acts as a sequence-specific endonuclease. This ribozyme has a homogeneous 5' end but a somewhat heterogeneous 3' end, as is typical of RNA synthesized by transcription in vitro. To produce a more homogeneous ribozyme for both structural and enzymological studies, a hammerhead ribozyme was inserted at the 3' end of the Tetrahymena ribozyme. During transcription the hammerhead moiety self-cleaves to produce the L-21 A Tetrahymena ribozyme, which ends at A410 with a 2',3'-cyclic phosphate terminus. The new ribozyme has endoribonuclease activity equivalent to that of L-21 Scal under conditions where binding of substrate is rate-limiting, as well as under conditions where chemical cleavage by guanosine is rate-limiting. However, the L-21 A has lost activity in oligo(C) disproportionation (e.g., 2 pC5----pC4 + pC6), consistent with the previous proposal that this reaction occurs predominantly through a covalent ribozyme-substrate intermediate involving the 3'-terminal hydroxyl group of the ribozyme. Formation of such an intermediate would be prevented by the 2',3'-cyclic phosphate terminus. Thus the L-21 A ribozyme has simplified enzymatic activity, being fully active as an endonuclease but blocked for disproportionation.

Animals↗
PubMed1991

Ribozyme-catalyzed and nonenzymatic reactions of phosphate diesters: rate effects upon substitution of sulfur for a nonbridging phosphoryl oxygen atom.

The L-21 ScaI ribozyme derived from the intervening sequence of Tetrahymena thermophila pre-rRNA catalyzes a guanosine-dependent endonuclease reaction that is analogous to the first step in self-splicing of this intervening sequence. We now describe pre-steady-state kinetic experiments, with sulfur substituting for the pro-RP (nonbridging) phosphoryl oxygen atom at the site of cleavage, that test aspects of a kinetic model proposed for the ribozyme reaction (Herschlag, D., & Cech, T. R. (1990) Biochemistry 29, 10159-10171). Thio substitution does not affect the reaction with subsaturating oligonucleotide substrate and saturating guanosine ((kcat/Km)S), consistent with the previous finding that binding of the oligonucleotide substrate limits this rate constant. In contrast, there is a significant decrease in the rate of single-turnover reactions of ribozyme-bound (i.e., saturating) oligonucleotide substrate upon thio substitution, with decreases of 2.3-fold for the reaction with guanosine ((kcat/Km)G) and 7-fold for hydrolysis [i.e., with solvent replacing guanosine; kc(-G)]. These "thio effects" are consistent with rate-limiting chemistry, as shown by comparison with model reactions. Nonenzymatic nucleophilic substitution reactions of the phosphate diester, methyl 2,4-dinitrophenyl phosphate monoanion, are slowed 4-11-fold by thio substitution for reactions with hydroxide ion, formate ion, fluoride ion, pyridine, and nicotinamide. In addition, we have confirmed that thio substitution has no effect on the nonenzymatic alkaline cleavage of RNA (Burgers, P. M. J., & Eckstein, F. (1979) Biochemistry 18, 592-596). Considering the strong preference of Mg2+ for binding to oxygen rather than sulfur, the modest thio effect on the chemical step of the ribozyme-catalyzed reaction and the absence of a thio effect on the equilibrium constant for binding of the oligonucleotide substrate suggest that the pro-RP oxygen atom is not coordinated to Mg2+ in the E.S complex or in the transition state. General implications of thio effects in enzymatic reactions of phosphate diesters are discussed.

Animals↗
PubMed1991

Inhibition of telomerase by G-quartet DNA structures.

The ends or telomeres of the linear chromosomes of eukaryotes are composed of tandem repeats of short DNA sequences, one strand being rich in guanine (G strand) and the complementary strand in cytosine. Telomere synthesis involves the addition of telomeric repeats to the G strand by telomere terminal transferase (telomerase). Telomeric G-strand DNAs from a variety of organisms adopt compact structures, the most stable of which is explained by the formation of G-quartets. Here we investigate the capacity of the different folded forms of telomeric DNA to serve as primers for the Oxytricha nova telomerase in vitro. Formation of the K(+)-stabilized G-quartet structure in a primer inhibits its use by telomerase. Furthermore, the octanucleotide T4G4, which does not fold, is a better primer than (T4G4)2, which can form a foldback structure. We conclude that telomerase does not require any folding of its DNA primer. Folding of telomeric DNA into G-quartet structures seems to influence the extent of telomere elongation in vitro and might therefore act as a negative regulator of elongation in vivo.

Animals↗