Biomedical subjects

T R Cech

Publications and source records attributed to T R Cech.

At least 55 records · Page 3Linked to original sources

PubMed1997

Three Ever Shorter Telomere (EST) genes are dispensable for in vitro yeast telomerase activity.

Telomerase is a specialized reverse transcriptase consisting of both RNA and protein components. Previous characterization of yeast telomerase function in vivo identified four EST (for ever shorter telomeres) genes that, when mutated, result in the phenotypes expected for a defect in telomerase. Consistent with this genetic prediction, the EST2 gene has recently been shown to encode the catalytic component of telomerase. Using an in vitro assay, we show here that telomerase activity is present in extracts prepared from yeast strains carrying est1-Delta, est3-Delta, and cdc13-2(est) mutations. Therefore, while these three genes are necessary for telomerase function in vivo, they do not encode components essential for core catalytic activity. When Est2p, the one EST gene product found to be essential for catalytic activity, was immunoprecipitated from extracts, the telomerase RNA subunit was also specifically precipitated, supporting the conclusion that these two components are in a stable complex.

Centrifugation, Density Gradient↗

PubMed1997

dGTP-dependent processivity and possible template switching of euplotes telomerase.

We have measured the processivity of telomeric DNA extension by Euplotes aediculatus telomerase at various concentrations of the nucleotide substrates dGTP and dTTP. The maximum processivity (approximately 3 repeats) was observed at approximately 100 microM of each dNTP. Processivity decreased as the dNTP concentrations were reduced and, surprisingly, as the concentration of dGTP was increased. Also, the characteristic banding pattern generated by telomerase extension of DNA primers shifted in response to changes in dGTP concentration. One pattern with 8 nt periodicity was predominant at dGTP concentrations </=16 microM, while at >/= 250 microM an 8 nt repeat pattern out-of-phase with the first was observed; at intermediate concentrations the two patterns coexisted. We propose that two different segments of the RNA subunit can serve as the template for repeat synthesis; nt 42-49 at low dGTP concentrations and nt 36-43 at high dGTP concentrations. An alternative model for the low dGTP pattern involves an internal pause site but no pause at the end of the template and is, therefore, considered less likely. Because the effects of dGTP on processivity and banding pattern appear to be distinct from nucleotide binding in the polymerase active site, we propose a second dGTP binding site involved in template selection and processivity.

Animals↗

PubMed1997

A second catalytic metal ion in group I ribozyme.

Although only a subset of protein enzymes depend on the presence of a metal ion for their catalytic function, all naturally occurring RNA enzymes require metal ions to stabilize their structure and for catalytic competence. In the self-splicing group I intron from Tetrahymena thermophila, several divalent metals can serve structural roles, but only Mg2+ and Mn2+ promote splice-site cleavage and exon ligation. A study of a ribozyme reaction analogous to 5'-splice-site cleavage by guanosine uncovered the first metal ion with a definitive role in catalysis. Substitution of the 3'-oxygen of the leaving group with sulphur resulted in a metal-specificity switch, indicating an interaction between the leaving group and the metal ion. Here we use 3'-(thioinosylyl)-(3'-->5')-uridine, IspU, as a substrate in a reaction that emulates exon ligation. Activity requires the addition of a thiophilic metal ion (Cd2+ or Mn2+), providing evidence for stabilization of the leaving group by a metal ion in that step of splicing. Based on the principle of microscopic reversibility, this metal ion activates the nucleophilic 3'-hydroxyl of guanosine in the first step of splicing, supporting the model of a two-metal-ion active site.

Animals↗

PubMed1997

Telomerase catalytic subunit homologs from fission yeast and human.

Catalytic protein subunits of telomerase from the ciliate Euplotes aediculatus and the yeast Saccharomyces cerevisiae contain reverse transcriptase motifs. Here the homologous genes from the fission yeast Schizosaccharomyces pombe and human are identified. Disruption of the S. pombe gene resulted in telomere shortening and senescence, and expression of mRNA from the human gene correlated with telomerase activity in cell lines. Sequence comparisons placed the telomerase proteins in the reverse transcriptase family but revealed hallmarks that distinguish them from retroviral and retrotransposon relatives. Thus, the proposed telomerase catalytic subunits are phylogenetically conserved and represent a deep branch in the evolution of reverse transcriptases.

Amino Acid Sequence↗

PubMed1997

Crystals by design: a strategy for crystallization of a ribozyme derived from the Tetrahymena group I intron.

Recently, the 2.8 A crystal structure of one domain of the self-splicing Tetrahymena group I intron was reported. Although it revealed much about RNA tertiary interactions, it contained only half of the active site. We have now designed a series of larger molecules that contain about 70% of the intron and all of the catalytic core. These RNAs were efficient in cleavage of a substrate RNA, consisting of the approximately 100 nucleotides from the 5' end of the intron, at a site corresponding to the 5' splice site. A sparse matrix was designed specifically for large RNAs and used to screen for preliminary crystallization conditions. Of the six RNAs initially tested, five were crystallized in this initial trial. Two of these crystals were further examined. The first diffracted X-rays to only approximately 16 A resolution, even when the crystal were very large. The second diffracted as high as 3.5 A, but the crystals were twinned and therefore unusable for structural studies. Site-specific mutagenesis was performed on the latter RNA to disrupt interactions that might have been responsible for the twinning. One of these mutant RNAs produced large, single, diffraction-quality crystals. The crystals belong to the tetragonal space group P42212 and have large unit cell dimensions, a=b=178 A and c=199 A. Thus, by variation of both sequence elements and crystallization conditions, crystals of a 247 nucleotide catalytic RNA were obtained.

Animals↗

PubMed1997

Effects of divalent metal ions on individual steps of the Tetrahymena ribozyme reaction.

The Tetrahymena thermophila L-21 ScaI ribozyme utilizes Mg2+ to catalyze a site-specific endonuclease reaction analogous to the first step of self-splicing. To better understand the contribution of Mg2+ to ribozyme activity, the Mg2+ concentration dependence of individual rate constants was examined at concentrations greater than those required for ribozyme folding (>2 mM; at 50 degrees C and pH 6.7). Analysis of metal ion inhibition of the chemical step of the reaction indicated that two Ca2+ ions compete with two Mg2+ ions involved in active site chemistry. These Mg2+ ions are bound tightly to the E.S complex (Kd < 2 mM). The rate constant for association of the oligoribonucleotide substrate (S) increased 12-fold from 2 to 100 mM Mg2+ and exhibited saturation behavior, consistent with a single Mg2+ ion involved in S association that binds to the free ribozyme with a Kd for Mg2+ of 15 mM. The preference for the divalent metal ion (Mg2+ congruent with Ca2+ > Ba2+ >> Sr2+) suggested that enhancing the rate constant of S association is not simply a function of ionic strength, but is due to a distinct metal ion binding site. Even though Ca2+ does not support reaction, the RNA substrate S was able to bind in the presence of Ca2+. Upon addition of Mg2+, S was cleaved without first dissociating. A model is proposed in which the inactive Ca2+ form of E.S is structurally equivalent to the open complex along the reaction pathway, which has the RNA substrate bound but not docked into the active site. Weaker binding of S in Ca2+ was shown to result from an increase in the rate constant of S dissociation, leading to the proposal that a tight Mg2+ binding site or sites in the E.S complex contribute to the strong binding of S. In summary, the data provide evidence for four functions for bound Mg2+ ions in the catalytic cycle: one increases the rate of RNA substrate binding, one or more decrease the rate of dissociation of S, and two are involved in the chemical step.

Animals↗

PubMed1997

Reverse transcriptase motifs in the catalytic subunit of telomerase.

Telomerase is a ribonucleoprotein enzyme essential for the replication of chromosome termini in most eukaryotes. Telomerase RNA components have been identified from many organisms, but no protein component has been demonstrated to catalyze telomeric DNA extension. Telomerase was purified from Euplotes aediculatus, a ciliated protozoan, and one of its proteins was partially sequenced by nanoelectrospray tandem mass spectrometry. Cloning and sequence analysis of the corresponding gene revealed that this 123-kilodalton protein (p123) contains reverse transcriptase motifs. A yeast (Saccharomyces cerevisiae) homolog was found and subsequently identified as EST2 (ever shorter telomeres), deletion of which had independently been shown to produce telomere defects. Introduction of single amino acid substitutions within the reverse transcriptase motifs of Est2 protein led to telomere shortening and senescence in yeast, indicating that these motifs are important for catalysis of telomere elongation in vivo. In vitro telomeric DNA extension occurred with extracts from wild-type yeast but not from est2 mutants or mutants deficient in telomerase RNA. Thus, the reverse transcriptase protein fold, previously known to be involved in retroviral replication and retrotransposition, is essential for normal chromosome telomere replication in diverse eukaryotes.

Amino Acid Sequence↗

PubMed1997

Regulation of telomere length and function by a Myb-domain protein in fission yeast.

Telomeres, the specialized nucleoprotein structures that comprise the ends of eukaryotic chromosomes, are essential for complete replication, and regulation of their length has been a focus of research on tumorigenesis. In the budding yeast Saccharomyces cerevisiae, the protein Rap1p binds to telomeric DNA and functions in the regulation of telomere length. A human telomere protein, hTRF (human TTAGGG repeat factor) binds the telomere sequence in vitro and localizes to telomeres cytologically, but its functions are not yet known. Here we use a genetic screen to identify a telomere protein in fission yeast, Taz1p (telomere-associated in Schizosaccharomyces pombe), that shares homology to the Myb proto-oncogene DNA-binding domain with hTRF. Disruption or deletion of the taz1+ gene causes a massive increase in telomere length. Taz1p is required for the repression of telomere-adjacent gene expression and for normal meiosis or sporulation. It may be a negative regulator of the telomere-replicating enzyme, telomerase, or may protect against activation of telomerase-independent pathways of telomere elongation.

Amino Acid Sequence↗

PubMed1997

Joining the two domains of a group I ribozyme to form the catalytic core.

Self-splicing group I introns, like other large catalytic RNAs, contain structural domains. Although the crystal structure of one of these domains has been determined by x-ray analysis, its connection to the other major domain that contains the guanosine-binding site has not been known. Site-directed mutagenesis and kinetic analysis of RNA splicing were used to identify a base triple in the conserved core of both a cyanobacterial (Anabaena) and a eukaryotic (Tetrahymena) group I intron. This long-range interaction connects a sequence adjacent to the guanosine-binding site with the domain implicated in coordinating the 5' splice site helix, and it thereby contributes to formation of the active site. The resulting five-strand junction, in which a short helix forms base triples with three separate strands in the Tetrahymena intron, reveals exceptionally dense packing of RNA.

Anabaena↗

PubMed1997

Telomerase and the chromosome end replication problem.

Telomerase, the enzyme that extends chromosomal DNA ends in most eukaryotes, contains essential RNA and protein subunits. We have been studying telomere replication in hypotrichous ciliates such as Euplotes aediculatus, which have numerous short macronuclear DNA molecules and therefore are highly enriched in telomeres and in telomerase. Cloning and sequencing genes for the RNA subunits from several ciliates revealed that telomerase RNAs with insignificant nucleotide sequence homology nevertheless form a common secondary structure. Affinity chromatography based on the sequence of the RNA subunit was used to purify the Euplotes telomerase as an active ribonucleoprotein enzyme. Two protein subunits, 123 kDa and 43 kDa, were identified. The finding of a yeast homologue to the 123 kDa subunit suggests that telomerase protein components may be much more highly conserved in evolution than the RNA subunits. The purified Euplotes telomerase has no activity with blunt-ended DNA primers, but instead requires a four to six nucleotide single-stranded 3' tail. This result supports a model for telomere replication in which other activities such as helicases or nucleases activate replicated DNA for extension by telomerase, a model that may be applicable to telomere replication in diverse eukaryotes.

Animals↗

PubMed1997

The anchor site of telomerase from Euplotes aediculatus revealed by photo-cross-linking to single- and double-stranded DNA primers.

Telomerase is a ribonucleoprotein enzyme that adds telomeric sequence repeats to the ends of linear chromosomes. In vitro, telomerase has been observed to add repeats to a DNA oligonucleotide primer in a processive manner, leading to the postulation of a DNA anchor site separate from the catalytic site of the enzyme. We have substituted photoreactive 5-iododeoxypyrimidines into the DNA oligonucleotide primer d(T4G4T4G4T4G2) and, upon irradiation, obtained cross-links with the anchor site of telomerase from Euplotes aediculatus nuclear extract. No cross-linking occurred with a primer having the same 5' end and a nontelomeric 3' end. These cross-links were shown to be between the DNA primer and (i) a protein moiety of approximately 130 kDa and (ii) U51-U52 of the telomerase RNA. The cross-linked primer could be extended by telomerase in the presence of [alpha-32P]dGTP, thus indicating that the 3' end was bound in the enzyme active site. The locations of the cross-links within the single-stranded primers were 20 to 22 nucleotides upstream of the 3' end, providing a measure of the length of DNA required to span the telomerase active and anchor sites. When the single-stranded primers are aligned with the G-rich strand of a Euplotes telomere, the cross-linked nucleotides correspond to the duplex region. Consistent with this finding, a cross-link to telomerase was obtained by substitution of 5-iododeoxycytidine into the CA strand of the duplex region of telomere analogs. We conclude that the anchor site in the approximately 130-kDa protein can bind duplex as well as single-stranded DNA, which may be critical for its function at chromosome ends. Quantitation of the processivity with single-stranded DNA primers and double-stranded primers with 3' tails showed that only 60% of the primer remains bound after each repeat addition.

Animals↗

PubMed1997

An RNA internal loop acts as a hinge to facilitate ribozyme folding and catalysis.

RNA molecules commonly consist of helical regions separated by internal loops, and in many cases these internal loops have been found to assume stable structures. We have examined the function and dynamics of an internal loop, J5/5a, that joins the two halves of the P4-P6 domain of the Tetrahymena self-splicing group I intron. P4-P6 RNAs with mutations in the J5/5a region showed nondenaturing gel electrophoretic mobilities and levels of Fe(II)-EDTA cleavage protection intermediate between those of wild-type RNA and a mutant incapable of folding into the native P4-P6 tertiary structure. Mutants with the least structured J5/5a loops behaved the most like wild-type P4-P6, and required smaller amounts of Mg2+ to rescue folding. The activity of reconstituted introns containing mutant P4-P6 RNAs correlated similarly with the nature of the J5/5a mutation. Our results suggest that, in solution, the P4-P6 RNA is in a two-state equilibrium between folded and unfolded states. We conclude that this internal loop mainly acts as a flexible hinge, allowing the coaxially stacked helical regions on either side of it to interact via specific tertiary contacts. To a lesser extent, the specific bases within the loop contribute to folding. Furthermore, it is crucial that the junction remain unstructured in the unfolded state. These conclusions cannot be derived from a simple examination of the P4-P6 crystal structure (Cate JH et al., 1996, Science 273:1678-1685), showing once again that structure determination must be supplemented with mutational and thermodynamic analysis to provide a complete picture of a folded macromolecule.

Base Sequence↗

PubMed1997

Mutagenesis and comparative sequence analysis of a base triple joining the two domains of group I ribozymes.

Tertiary interactions are important in the higher-order folding of catalytic RNAs. Recently, a base triple, joining the two major domains of the catalytic core, was determined in group I introns from the cyanobacterium Anabaena PCC7120 and the eukaryote Tetrahymena thermophila. This base triple involves the fifth base pair of P4 and the fifth base of the single-stranded region J8/7. We made base pair and single-nucleotide substitutions in the fifth base pair of P4, a G-C in the wild-type Anabaena intron, and tested them for self-splicing activity. The results suggest a hydrogen bonding model in which only the C of the base pair interacts directly with the fifth base of J8/7. Comparative sequence analysis was used to determine the different combinations of base triples that occur in approximately 450 natural group I introns identified to date. About 94% of the base triples analyzed are compatible with the proposed hydrogen bonding model. Disrupting this base triple in the Tetrahymena intron resulted in the disappearance of splicing intermediates (intron 3' exon and 5' exon), even though the first step of splicing was not affected. Restoration of the base triple by a compensatory mutation reverted the intermediates to wild-type levels. These results suggest that disruption of the base triple increases the rate of the second step of splicing or of a conformational change preceding the second step. Repositioning of the base triple to form a new set of interactions may be required for the second step of splicing.

Anabaena↗

PubMed1997

Polyadenylation of telomerase RNA in budding yeast.

Telomerase RNA is a subunit of a stable ribonucleoprotein particle required for telomere replication. We find that, at steady state, 5-10% of the telomerase RNA in Saccharomyces cerevisiae and Kluyveromyces lactis contains a poly(A) tail of about 80 nt. In S. cerevisiae, the poly(A)+ fraction quickly disappeared when a conditional pap1 or rna15 mutant was shifted to the nonpermissive temperature, indicating that polyadenylation is accomplished by the same machinery that polyadenylates mRNAs. Potential cis-acting polyadenylation elements were identified in the telomerase RNA sequence; when they were mutagenized, the polyadenylation pattern shifted, but was not eliminated. The corresponding mutants displayed wild-type growth. By putting the RNA under the control of an inducible promoter, we were able to show that synthesis of the poly(A)+ RNA precedes that of the poly(A)- fraction. This supports, but does not prove, a model in which all telomerase RNA is first polyadenylated and then rapidly processed to give the stable poly(A)-form. Cell cycle arrest experiments showed an increase in the poly(A)+ form between G1 and S phase, consistent with an induction of telomerase RNA transcription at the time of DNA replication.

Base Sequence↗

PubMed1997

Telomerase is a true reverse transcriptase. A review.

Synthesis of telomeric repeats at chromosome ends requires telomerase, a ribonucleoprotein enzyme. The RNA subunit, which contains the template for DNA synthesis, has been identified in many organisms. Recently, the protein subunit that catalyzes telomeric DNA extension has also been identified in Euplotes aediculatus and Saccharomyces cerevisiae. It has sequence and functional characteristics of a reverse transcriptase related to retrotransposon and retroviral reverse transcriptases, so this new family of telomerase subunits has been named TRT (Telomerase Reverse Transcriptase). We find it remarkable that the same type of protein structure required for retroviral replication is now seen to be essential for normal chromosome telomere replication in diverse eukaryotes.

Animals↗

PubMed1996

The anticodon is the signal sequence for mitochondrial import of glutamine tRNA in Tetrahymena.

The import of nuclear-encoded RNAs into mitochondria is required for proper mitochondrial function in most organisms. However, the mechanisms used to achieve RNA import are largely unknown. In particular, the RNA elements that direct import have not been identified in any organism. In Tetrahymena, only one of three nuclear-encoded glutamine accepting tRNAs is imported into mitochondria. We transform Tetrahymena with marked glutamine tRNAs and quantitate their level of accumulation in mitochondria. Of several isostructural nucleotide substitutions tested, alteration of the anticodon sequence uniquely abolishes import. Furthermore, substitution of a single anticodon nucleotide (UUA-->UUG) confers import on a normally nonimported glutamine tRNA. Thus, the anticodon functions as a mitochondrial localization signal and is both necessary and sufficient for tRNA import. Given the prior evidence that neither the cytoplasmic nor the mitochondrial glutaminyl-tRNA synthetase distinguishes between the imported and nonimported glutamine tRNAs with respect to aminoacylation, we propose that some mitochondrial import factor distinct from a synthetase recognizes the anticodon of the imported glutamine tRNA.

Amino Acyl-tRNA Synthetases↗

PubMed1996

Purification of telomerase from Euplotes aediculatus: requirement of a primer 3' overhang.

Telomerase is a ribonucleoprotein enzyme that uses its internal RNA moiety as a template for synthesis of telomeric repeats at chromosome ends. Here we report the purification of telomerase from Euplotes aediculatus by affinity chromatography with antisense 2'-O-methyl oligonucleotides, a method that was developed for small nuclear ribonucleoprotein particles (snRNPs). Elution of bound ribonucleoprotein from the antisense oligonucleotide under nondenaturing conditions was achieved by a novel approach, using a displacement oligonucleotide. Polypeptides of 120 kDa and 43 kDa (a doublet) copurify with the active telomerase and appear stoichiometric with telomerase RNA. A simple model for DNA end replication predicts that after semiconservative DNA replication, telomerase will extend the newly synthesized, blunt-ended leading strand. We show that purified Euplotes telomerase has no activity with blunt-ended primers. Instead, efficient extension requires 4 to 6 single-stranded nucleotides at the 3' end. Therefore, this model predicts the existence of other activities such as helicases or nucleases that generate a single-stranded 3' end from a blunt end, thus activating the end for telomerase extension.

Animals↗

PubMed1996

Conserved thermochemistry of guanosine nucleophile binding for structurally distinct group I ribozymes.

We report thermodynamic values for binding of the guanosine nucleophile to the ribozyme derived from the Anabaena group I intron, and find that they are similar to those measured previously for the structurally distinct Tetrahymena ribozyme. The free energy of binding guanosine 5'-monophosphate (pG) at 30 degrees C is similar for the two ribozymes. The delta(H)degrees' and delta(S)degrees' for pG binding to the Anabaena ribozyme--RNA substrate complex (E x S) are 3.4 +/- 4 kcal/mol and 27 +/- 10 e.u., respectively. The negligible enthalpic contribution and positive entropy change were found previously for the Tetrahymena ribozyme, and are considered remarkable for a hydrogen-bonding interaction between a nucleotide and a nucleic acid. These thermodynamic values may reflect conformational changes or water release upon pG binding that are comparable for the two ribozymes. In addition, the apparent chemical steps of the two ribozyme reactions share similar activation energies and a positive deltaS++. It now appears that such thermochemical values for guanosine binding and activation may be intrinsic properties of the group I intron catalytic center.

Anabaena↗