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T R Cech

Publications and source records attributed to T R Cech.

At least 19 recordsLinked to original sources
PubMed2002

Ribozymes, the first 20 years.

In 1982 we reported the first catalytic RNA or ribozyme: the self-splicing intron of the Tetrahymena pre-rRNA. Additional examples of natural ribozymes were soon found, and research in the field focused on their enzymic mechanism and secondary and tertiary structure. Ribozymes identified through in vitro selection extended the repertoire of RNA catalysis. Two directions of current and future interest are the determination of atomic-resolution structures of large ribozymes by X-ray crystallography and the structural and mechanistic analysis of complexes of ribozymes with protein facilitators of their activity.

Introns↗
PubMed2001

A stem-loop of Tetrahymena telomerase RNA distant from the template potentiates RNA folding and telomerase activity.

The ribonucleoprotein enzyme telomerase adds telomeric repeats to the ends of linear chromosomes. The Tetrahymena telomerase reverse transcriptase (TERT) protein and the telomerase RNA can be reconstituted into an active complex in vitro in rabbit reticulocyte lysates. We have probed the structure of the telomerase RNA in the reconstituted complex with RNases T1 and V1. Upon TERT binding to the RNA, sites of both protection and enhancement of cleavage were observed, suggesting potential protein-binding sites and conformational changes in the RNA. Especially prominent was a large region of RNase V1 protection in stem-loop IV. A number of loop IV mutants still bound TERT but showed drastic decreases in the level of telomerase activity and the loss of protein-dependent folding of the pseudoknot region of the telomerase RNA. The telomerase activity defect and the misfolding of the pseudoknot were partially separable, leading to the proposal of two functions for stem-loop IV: to aid in the folding of the pseudoknot and to function more directly in the active site of telomerase. Thus an RNA element far from the template makes a major contribution to Tetrahymena telomerase enzyme activity.

Animals↗
PubMed2001

Pot1, the putative telomere end-binding protein in fission yeast and humans.

Telomere proteins from ciliated protozoa bind to the single-stranded G-rich DNA extensions at the ends of macronuclear chromosomes. We have now identified homologous proteins in fission yeast and in humans. These Pot1 (protection of telomeres) proteins each bind the G-rich strand of their own telomeric repeat sequence, consistent with a direct role in protecting chromosome ends. Deletion of the fission yeast pot1+ gene has an immediate effect on chromosome stability, causing rapid loss of telomeric DNA and chromosome circularization. It now appears that the protein that caps the ends of chromosomes is widely dispersed throughout the eukaryotic kingdom.

Amino Acid Sequence↗
PubMed2001

Structural basis of the enhanced stability of a mutant ribozyme domain and a detailed view of RNA--solvent interactions.

BACKGROUND: The structure of P4-P6, a 160 nucleotide domain of the self-splicing Tetrahymena thermophila intron, was solved previously. Mutants of the P4-P6 RNA that form a more stable tertiary structure in solution were recently isolated by successive rounds of in vitro selection and amplification. RESULTS: We show that a single-site mutant (Delta C209) possessing greater tertiary stability than wild-type P4-P6 also crystallizes much more rapidly and under a wider variety of conditions. The crystal structure provides a satisfying explanation for the increased stability of the mutant; the deletion of C209 allows the adjacent bulged adenine to enter the P4 helix and form an A-G base pair, presumably attenuating the conformational flexibility of the helix. The structure of another mutant (Delta A210) was also solved and supports this interpretation. The crystals of Delta C209 diffract to a higher resolution limit than those of wild-type RNA (2.25 A versus 2.8 A), allowing assignment of innersphere and outersphere coordination contacts for 27 magnesium ions. Structural analysis reveals an intricate solvent scaffold with a preponderance of ordered water molecules on the inside rather than the surface of the folded RNA domain. CONCLUSIONS: In vitro evolution facilitated the identification of a highly stable, structurally homogeneous mutant RNA that was readily crystallizable. Analysis of the structure suggests that improving RNA secondary structure can stabilize tertiary structure and perhaps promote crystallization. In addition, the higher resolution model provides new details of metal ion-RNA interactions and identifies a core of ordered water molecules that may be integral to RNA tertiary structure formation.

Animals↗
PubMed2001

An early transition state for folding of the P4-P6 RNA domain.

Tertiary folding of the 160-nt P4-P6 domain of the Tetrahymena group I intron RNA involves burying of substantial surface area, providing a model for the folding of other large RNA domains involved in catalysis. Stopped-flow fluorescence was used to monitor the Mg2+-induced tertiary folding of pyrene-labeled P4-P6. At 35 degrees C with [Mg2+] approximately 10 mM, P4-P6 folds on the tens of milliseconds timescale with k(obs) = 15-31 s(-1). From these values, an activation free energy deltaG(double dagger) of approximately 8-16 kcal/mol is calculated, where the large range for deltaG(double dagger) arises from uncertainty in the pre-exponential factor relating k(obs) and delta G(double dagger). The folding rates of six mutant P4-P6 RNAs were measured and found to be similar to that of the wild-type RNA, in spite of significant thermodynamic destabilization or stabilization. The ratios of the kinetic and thermodynamic free energy changes phi = delta deltaG(double dagger)/delta deltaG(o') are approximately 0, implying a folding transition state in which most of the native-state tertiary contacts are not yet formed (an early folding transition state). The k(obs) depends on the Mg2+ concentration, and the initial slope of k(obs) versus [Mg2+] suggests that only approximately 1 Mg2+ ion is bound in the rate-limiting folding step. This is consistent with an early folding transition state, because folded P4-P6 binds many Mg2+ ions. The observation of a substantial deltaG(double dagger) despite an early folding transition state suggests that a simple two-state folding diagram for Mg2+-induced P4-P6 folding is incomplete. Our kinetic data are some of the first to provide quantitative values for an activation barrier and location of a transition state for tertiary folding of an RNA domain.

Animals↗
PubMed2000

Euplotes telomerase contains an La motif protein produced by apparent translational frameshifting.

Telomerase is the ribonucleoprotein enzyme responsible for the replication of chromosome ends in most eukaryotes. In the ciliate Euplotes aediculatus, the protein p43 biochemically co-purifies with active telomerase and appears to be stoichiometric with both the RNA and the catalytic protein subunit of this telomerase complex. Here we describe cloning of the gene for p43 and present evidence that it is an authentic component of the telomerase holoenzyme. Comparison of the nucleotide sequence of the cloned gene with peptide sequences of the protein suggests that production of full-length p43 relies on a programmed ribosomal frameshift, an extremely rare translational mechanism. Anti-p43 antibodies immunodeplete telomerase RNA and telomerase activity from E.aediculatus nuclear extracts, indicating that the vast majority of mature telomerase complexes in the cell are associated with p43. The sequence of p43 reveals similarity to the La autoantigen, an RNA-binding protein involved in maturation of RNA polymerase III transcripts, and recombinant p43 binds telomerase RNA in vitro. By analogy to other La proteins, p43 may function in chaperoning the assembly and/or facilitating nuclear retention of telomerase.

Amino Acid Motifs↗
PubMed2000

Multiple folding pathways for the P4-P6 RNA domain.

We recently described site-specific pyrene labeling of RNA to monitor Mg(2+)-dependent equilibrium formation of tertiary structure. Here we extend these studies to follow the folding kinetics of the 160-nucleotide P4-P6 domain of the Tetrahymena group I intron RNA, using stopped-flow fluorescence with approximately 1 ms time resolution. Pyrene-labeled P4-P6 was prepared using a new phosphoramidite that allows high-yield automated synthesis of oligoribonucleotides with pyrene incorporated at a specific 2'-amino-2'-deoxyuridine residue. P4-P6 forms its higher-order tertiary structure rapidly, with k(obs) = 15-31 s(-1) (t(1/2) approximately 20-50 ms) at 35 degrees C and [Mg(2+)] approximately 10 mM in Tris-borate (TB) buffer. The folding rate increases strongly with temperature from 4 to 45 degrees C, demonstrating a large activation enthalpy DeltaH(double dagger) approximately 26 kcal/mol; the activation entropy DeltaS(double dagger) is large and positive. In low ionic strength 10 mM sodium cacodylate buffer at 35 degrees C, a slow (t(1/2) approximately 1 s) folding component is also observed. The folding kinetics are both ionic strength- and temperature-dependent; the slow phase vanishes upon increasing [Na(+)] in the cacodylate buffer, and the kinetics switch completely from fast at 30 degrees C to slow at 40 degrees C. Using synchrotron hydroxyl radical footprinting, we confirm that fluorescence monitors the same kinetic events as hydroxyl radical cleavage, and we show that the previously reported slow P4-P6 folding kinetics apply only to low ionic strength conditions. One model to explain the fast and slow folding kinetics postulates that some tertiary interactions are present even without Mg(2+) in the initial state. The fast kinetic phase reflects folding that is facilitated by these interactions, whereas the slow kinetics are observed when these interactions are disrupted at lower ionic strength and higher temperature.

Animals↗
PubMed2000

Structural biology. The ribosome is a ribozyme.

Ribosomes, the cellular factories that manufacture proteins, contain both RNA and protein, but exactly how all of the different ribosomal components contribute to protein synthesis is still not clear. Now, as Thomas Cech explains in his Perspective, atomic resolution of the structure of the large ribosomal subunit reveals that, as predicted by those convinced of a prebiotic RNA world, RNA is the catalytic component with proteins being the structural units that support and stabilize it (Ban et al., Nissen et al., Muth et al.).

Adenine↗
PubMed2000

A mutant of Tetrahymena telomerase reverse transcriptase with increased processivity.

The protein catalytic subunit of telomerase (TERT) is a reverse transcriptase (RT) that utilizes an internal RNA molecule as a template for the extension of chromosomal DNA ends. In all retroviral RTs there is a conserved tyrosine two amino acids preceding the catalytic aspartic acids in motif C, a motif that is critical for catalysis. In TERTs, however, this position is a leucine, valine, or phenylalanine. We developed and characterized a robust in vitro reconstitution system for Tetrahymena telomerase and tested the effects of amino acid substitutions on activity. Substitution of the retroviral-like tyrosine in motif C did not change overall enzymatic activity but increased processivity. This increase in processivity correlated with an increased affinity for telomeric DNA primer. Substitution of an alanine did not increase processivity, while substitution of a phenylalanine had an intermediate effect. The data suggest that this amino acid is involved in interactions with the primer in telomerase as in other RTs, and show that mutating an amino acid to that conserved in retroviral RTs makes telomerase more closely resemble these other RTs.

Amino Acid Motifs↗
PubMed2000

Analysis of telomerase catalytic subunit mutants in vivo and in vitro in Schizosaccharomycespombe.

The chromosome end-replicating enzyme telomerase is composed of a template-containing RNA subunit, a reverse transcriptase (TERT), and additional proteins. The importance of conserved amino acid residues in Trt1p, the TERT of Schizosaccharomyces pombe, was tested. Mutation to alanine of the proposed catalytic aspartates in reverse transcriptase motifs A and C and of conserved amino acids in motifs 1 and B' resulted in defective growth, progressive loss of telomeric DNA, and loss of detectable telomerase enzymatic activity in vitro. Mutation of the phenylalanine (F) in the conserved FYxTE of telomerase-specific motif T had no phenotype in vivo or in vitro whereas mutation of a conserved amino acid in RT motif 2 had an intermediate effect. In addition to identifying single amino acids of TERT required for telomere maintenance in the fission yeast, this work provides useful tools for S. pombe telomerase research: a functional epitope-tagged version of Trt1p that allows detection of the protein even in crude cellular extracts, and a convenient and robust in vitro enzymatic activity assay based on immunopurification of telomerase.

Amino Acid Motifs↗
PubMed2000

Telomerase RNA bound by protein motifs specific to telomerase reverse transcriptase.

Telomerase reverse transcriptase (TERT) differs from many other reverse transcriptases in that it remains stably associated with its template-containing RNA subunit. Elements of TERT involved in binding the RNA subunit have now been identified by mutagenesis and in vitro reconstitution of the Tetrahymena ribonucleoprotein complex. Mutations in the reverse transcriptase motifs of TERT reduced activity as expected but did not greatly reduce its binding to the telomerase RNA. In contrast, all mutations in the T and CP motifs dramatically reduced RNA binding. We therefore suggest that the T and CP motifs of TERT function to hold on to the telomerase RNA, leaving the RNA template region free to translocate through the RT domain.

Amino Acid Sequence↗
PubMed2000

Protection of telomeres by the Ku protein in fission yeast.

Schizosaccharomyces pombe cells survive loss of telomeres by a unique pathway of chromosome circularization. Factors potentially involved in this survival mechanism include the heterodimeric Ku protein and ligase IV, both of which are involved in the repair of DNA double-strand breaks in mammalian cells. Furthermore, Ku plays a role in telomere maintenance as well as in DNA double-strand break repair in Saccharomyces cerevisiae. We have identified Ku and ligase IV homologues in S. pombe and analyzed their functions during normal growth and in cells undergoing senescence. In the absence of either a Ku subunit (pku70(+)) or ligase IV (lig4(+)), nonhomologous DNA end-joining was severely reduced. Lack of functional Ku led to shorter but stable telomeres and caused striking rearrangements of telomere-associated sequences, indicating a function for Ku in inhibiting recombinational activities near chromosome ends. In contrast to S. cerevisiae, concurrent deletion of pku70(+) and the gene for the catalytic subunit of telomerase (trt1(+)) was not lethal, allowing for the first time the dissection of the roles of Ku during senescence. Our results support a model in which Ku protects chromosome termini from nucleolytic and recombinational activities but is not involved in the formation of chromosome end fusions during senescence. The conclusion that nonhomologous end-joining is not required for chromosome circularization was further supported by analysis of survivors in strains lacking the genes for both trt1(+) and lig4(+).

Antigens, Nuclear↗
PubMed1999

Self-splicing of the Tetrahymena intron from mRNA in mammalian cells.

The Tetrahymena pre-rRNA self-splicing intron is shown to function in the unnatural context of an mRNA transcribed by RNA polymerase II in mammalian cells. Mutational analysis supports the conclusion that splicing in cells occurs by the same RNA-catalyzed mechanism established for splicing in vitro. Insertion of the intron at five positions spanning the luciferase open reading frame revealed 10-fold differences in accumulation of ligated exons and in luciferase activity; thus, the intron self-splices in many exon contexts, but the context can have a significant effect on activity. In addition, even the best self-splicing constructs, which produced half as much mRNA as did an uninterrupted luciferase gene, gave approximately 100-fold less luciferase enzyme activity, revealing an unexpected discontinuity between mRNA production and translation in cells. The finding that production of accurately spliced mRNA in cells does not guarantee a corresponding level of protein production is surprising, and may have implications for the development of trans-splicing ribozymes as therapeutics.

Amino Acid Substitution↗
PubMed1999

Essential functions of amino-terminal domains in the yeast telomerase catalytic subunit revealed by selection for viable mutants.

Telomerase is a ribonucleoprotein complex that adds telomeric DNA repeats to the ends of most eukaryotic chromosomes. The reverse transcriptase subunit of telomerase (TERT) differs from retroviral reverse transcriptases in having a long basic amino-terminal extension. We made a large library containing random mutations in the amino terminus of the EST2 gene, which encodes the Saccharomyces cerevisiae TERT, and selected functional alleles by their ability to rescue senescence of telomerase-negative cells. Through analysis of 265 mutations, the amino terminus of Est2p was found to contain at least four essential regions. This domain structure was verified by a combination of deletion and alanine-block mutations. Mutations within two essential domains of the protein reduced RNA binding, suggesting that the amino terminus of Est2p makes important contacts with the intrinsic RNA component of telomerase. A mutant close to the amino terminus retained RNA binding and in vitro enzymatic activity but was defective in vivo, suggesting a role in interaction with other macromolecular components of telomerase.

Alanine↗
PubMed1999

RNA tertiary folding monitored by fluorescence of covalently attached pyrene.

The pathways by which large RNAs adopt tertiary structure are just beginning to be explored, and new methods that reveal RNA folding are highly desirable. Here we report an assay for RNA tertiary folding in which the fluorescence of a covalently incorporated chromophore is monitored. Folding of the 160-nucleotide Tetrahymena group I intron P4-P6 domain was used as a test system. Guided by the P4-P6 X-ray crystal structure, we chose a nucleotide (U107) for which derivatization at the 2'-position should not perturb the folded conformation. A 15-mer RNA oligonucleotide with a 2'-amino substitution at U107 was derivatized with a pyrene chromophore on a variable-length tether, and then ligated to the remainder of P4-P6, providing a site-specifically pyrene-labeled P4-P6 derivative. Upon titration of the pyrene-derivatized P4-P6 with Mg(2+), the equilibrium fluorescence intensity reversibly increased several-fold, as expected if the probe's chemical microenvironment changes as the RNA to which it is attached folds. The concentration and specificity of divalent ions required to induce the fluorescence change (Mg(2+) approximately Ca(2+) > Sr(2+)) correlated well with biochemical folding assays that involve nondenaturing gel electrophoresis. Furthermore, mutations in P4-P6 remote from the chromophore that shifted the Mg(2+) folding requirement on nondenaturing gels also affected in a predictable way the Mg(2+) requirement for the fluorescence increase. Initial stopped-flow studies with millisecond time resolution suggest that this fluorescence method will be useful for following the kinetics of P4-P6 tertiary folding. We conclude that a single site-specifically tethered chromophore can report the formation of global structure of a large RNA molecule, allowing one to monitor both the equilibrium progress and the real-time kinetics of RNA tertiary folding.

Animals↗
PubMed1999

Saccharomyces cerevisiae telomerase is an Sm small nuclear ribonucleoprotein particle.

Activation of the chromosome end-replicating enzyme telomerase can greatly extend the lifespan of normal human cells and is associated with most human cancers. In all eukaryotes examined, telomerase has an RNA subunit, a conserved reverse transcriptase subunit and additional proteins, but little is known about the assembly of these components. Here we show that the Saccharomyces cerevisiae telomerase RNA has a 5'-2,2,7-trimethylguanosine (TMG) cap and a binding site for the Sm proteins, both hallmarks of small nuclear ribonucleoprotein particles (snRNPs) that are involved in nuclear messenger RNA splicing. Immunoprecipitation of telomerase from yeast extracts shows that Sm proteins are assembled on the RNA and that most or all of the telomerase activity is associated with the Sm-containing complex. These data support a model in which telomerase RNA is transcribed by RNA polymerase II and 7-methylguanosine-capped, binds the seven Sm proteins, becomes TMG-capped and picks up the other protein subunits. We conclude that the functions of snRNPs assembled by this pathway are not restricted to RNA processing, but also include chromosome telomere replication.

Autoantigens↗