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T R Cech

Publications and source records attributed to T R Cech.

At least 19 recordsLinked to original sources
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

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↗
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↗
PubMed1996

Crystal structure of a group I ribozyme domain: principles of RNA packing.

Group I self-splicing introns catalyze their own excision from precursor RNAs by way of a two-step transesterification reaction. The catalytic core of these ribozymes is formed by two structural domains. The 2.8-angstrom crystal structure of one of these, the P4-P6 domain of the Tetrahymena thermophila intron, is described. In the 160-nucleotide domain, a sharp bend allows stacked helices of the conserved core to pack alongside helices of an adjacent region. Two specific long-range interactions clamp the two halves of the domain together: a two-Mg2+-coordinated adenosine-rich corkscrew plugs into the minor groove of a helix, and a GAAA hairpin loop binds to a conserved 11-nucleotide internal loop. Metal- and ribose-mediated backbone contacts further stabilize the close side-by-side helical packing. The structure indicates the extent of RNA packing required for the function of large ribozymes, the spliceosome, and the ribosome.

Adenine↗
PubMed1996

RNA tertiary structure mediation by adenosine platforms.

The crystal structure of a group I intron domain reveals an unexpected motif that mediates both intra- and intermolecular interactions. At three separate locations in the 160-nucleotide domain, adjacent adenosines in the sequence lie side-by-side and form a pseudo-base pair within a helix. This adenosine platform opens the minor groove for base stacking or base pairing with nucleotides from a noncontiguous RNA strand. The platform motif has a distinctive chemical modification signature that may enable its detection in other structured RNAs. The ability of this motif to facilitate higher order folding provides one explanation for the abundance of adenosine residues in internal loops of many RNAs.

Adenosine↗
PubMed1996

Mutations in the Tetrahymena ribozyme internal guide sequence: effects on docking of the P1 helix into the catalytic core and correlation with catalytic activity.

Binding of substrate by the ribozyme derived from the self-splicing intron of Tetrahymena thermophila involves at least two steps. In the first step, base pairing between the ribozyme internal guide sequence (IGS) and the substrate forms a helical duplex (P1). Through specific tertiary interactions between P1 and the ribozyme core, P1 is then docked into the ribozyme active site. We have investigated the effects of compensatory mutations in positions 2-6 of the P1 helix on docking of P1 into the ribozyme core. Equilibrium binding of matching oligonucleotides by catalytically active IGS mutant ribozymes was evaluated by gel-shift analysis. While the strength of base pairing changed with base composition as expected, the strength of tertiary interactions between P1 and the ribozyme core was not affected by the P1 mutations. These results support a model in which efficient docking of P1 is determined by P1 structure and the presence of a conserved G-U pair. Determination of the rate of dissociation of matching oligonucleotides from each ribozyme revealed that mutations in the IGS change the tightness of binding by increasing or decreasing the dissociation rate. Surprisingly, dissociation rates determined in this fashion were 20-900-fold less than the values of the multiple-turnover rate constant for these ribozymes, initially suggesting that turnover did not require product dissociation. A more detailed analysis for the wild-type ribozyme defined two distinct product dissociation rates. The slower rate equaled that determined under the conditions used for the equilibrium binding studies. The weighted average of the two dissociation rates equaled the multiple-turnover rate constant. These results are explained by a model in which ribozyme preparations consist of two ribozyme conformers: one with tight docking of P1 and another with weaker docking of P1.

Animals↗
PubMed1996

Minor-groove recognition of double-stranded RNA by the double-stranded RNA-binding domain from the RNA-activated protein kinase PKR.

The human double-stranded RNA- (dsRNA) activated protein kinase (PKR) has a dsRNA-binding domain (dsRBD) that contains two tandem copies of the dsRNA-binding motif (dsRBM). The minimal-length polypeptide required to bind dsRNA contains both dsRBMs, as determined by mobility-shift and filter-binding assays. Mobility-shift experiments indicate binding requires a minimum of 16 base pairs of dsRNA, while a minimal-length site for saturation of longer RNAs is 11 base pairs. Bulge defects in the helix disfavor binding, and single-stranded tails do not strongly influence the dsRNA length requirement. These polypeptides do not bind an RNA-DNA hybrid duplex or dsDNA as judged by either mobility-shift or competition experiments, suggesting 2'-OH contacts on both strands of the duplex stabilize binding. Related experiments on chimeric duplexes in which specific sets of 2'-OHs are substituted with 2'-H or 2'-OCH3 reveal that the 2'-OHs required for binding are located along the entire 11 basepair site. These results are supported by Fe(II) EDTA footprinting experiments that show protein-dependent protection of the minor groove of dsRNA. The dependence of dsRNA-protein binding on salt concentration suggests that only one ionic contact is made between the protein and dsRNA phosphate backbone and that at physiological salt concentrations 90% of the free energy of binding is nonelectrostatic. Thus, the specificity of PKR for dsRNA over RNA-DNA hybrids and dsDNA is largely due to molecular recognition of a network of 2'-OHs involving both strands of dsRNA and present along the entire 11 base-pair site.

Amino Acid Sequence↗
PubMed1996

Mitochondrial import of only one of three nuclear-encoded glutamine tRNAs in Tetrahymena thermophila.

The mitochondrial genome of Tetrahymena does not appear to encode enough tRNAs to perform mitochondrial protein synthesis. It has therefore been proposed that nuclear-encoded tRNAs are imported into the mitochondria. T.thermophila has three major glutamine tRNAs: tRNA(Gln)(UUG), tRNA(Gln)(UUA) and tRNA(Gln)(CUA). Each of these tRNAs functions in cytosolic translation. However, due to differences between the Tetrahymena nuclear and mitochondrial genetic codes, only tRNA(Gln)(UUG) has the capacity to function in mitochondrial translation as well. Here we show that approximately 10-20% of the cellular complement of tRNA(Gln)(UUG) is present in mitochondrial RNA fractions, compared with 1% or less for the other two glutamine tRNAs. Furthermore, this glutamine tRNA is encoded only by a family of nuclear genes, the sequences of several of which are presented. Finally, when marked versions of tRNA(Gln)(UUG) and tRNA(Gln)(UUA) flanked by identical sequences are expressed in the macronucleus, only the former undergoes mitochondrial import; thus sequences within tRNA(Gln)(UUG) direct import. Because tRNA(Gln)(UUG) is a constituent of mitochondrial RNA fractions and is encoded only by nuclear genes, and because ectopically expressed tRNA(Gln)(UUG) fractionates with mitochondria like its endogenous counterpart, we conclude that it is an imported tRNA in T.thermophila.

Animals↗
PubMed1996

Conformational switches involved in orchestrating the successive steps of group I RNA splicing.

Group I introns possess a conserved guanosine residue at their 3' end, termed omegaG, that, in the case of the Tetrahymena pre-rRNA, is a major determinant of the second step of splicing. We examined the role of omegaG in self-splicing of the 249-residue group I intron of the Anabaena PCC7120 tRNAleu precursor. Contrary to observations with the Tetrahymena pre-rRNA intron, a mutation that places an adenosine residue at the omega position did not have a severe effect on the second step of splicing; neither 3' splice-site selection nor the rate of the second step was altered. The first step of splicing, however, was now readily reversed. This unexpected effect also resulted from a mutation that altered the nucleoside specificity of the intronic guanosine-binding site. The theme common to these mutations is that reversal of the first step of splicing results when there is not a strong interaction between the guanosine-binding site and the omega residue. This suggests that a major role of omegaG is to compete with the exogenous guanosine molecule added to the intron in the first step of splicing for the single guanosine-binding site of the intron. From these data, we are able to extend the mechanism for the self-splicing reaction of this intron by proposing two distinct conformational changes between the first and second steps of the splicing. The first of these is the exchange of the exogenous nucleoside for the omega nucleoside. This is the equilibrium that we can perturb by mutations at either the omega position or the guanosine-binding site. An additional conformational change then fully activates the intron for the second step of splicing.

Anabaena↗
PubMed1996

Exocyclic amine of the conserved G.U pair at the cleavage site of the Tetrahymena ribozyme contributes to 5'-splice site selection and transition state stabilization.

A phylogenetically conserved guanine.uracil (G.U) pair defines the 5'-exon/intron boundary of precursor RNAs containing group I introns. In this wobble base pair, the G forms two hydrogen bonds with U in a base pairing geometry shifted from that of a canonical Watson-Crick pair. On the basis of thermodynamic measurements of synthetic base pair analogs (inosine, diaminopurine riboside, guanosine, or adenosine paired with U, C, or isocytidine) in place of the G.U pair, we have previously reported that the N2 exocyclic amine of the G is important for docking the 5'-exon into the active site of the Tetrahymena ribozyme [Strobel, S. A., & Cech, T. R. (1995) Science 267, 675-679]. Here we describe kinetic characterization of ribozyme-substrate combinations containing the same series of analogs. By measuring the rate constants of 5'-exon miscleavage (cleavage at incorrect phosphates), we demonstrate that the 5'-exon/intron boundary is primarily defined by the exocyclic amine of the G. The amine makes its contribution (2.5 kcal.mol-1) in the context of all three wobble pairs tested but fails to make a significant contribution (< 0.8 kcal.mol-1) when presented in a Watson-Crick base pairing geometry. We also demonstrate that the exocyclic amine makes a modest contribution to chemical transition state stabilization (1.0 kcal.mol-1 relative to an inosine-U pair). The majority of this transition state contribution (0.7 kcal.mol-1) is independent of that contributed by the 2'-hydroxyl of the neighboring U. This argues against the model in which substantial transition state stabilization is derived from a water molecule bridging between the exocyclic amine of G and the 2'-hydroxyl of U. Instead it suggests that the tertiary interaction between the exocyclic amine and its hydrogen bonding partner in the active site is slightly improved during the chemical transition. We conclude that the exocyclic amine of G is the primary contributor to many characteristics of reactivity that have been ascribed to the conserved G.U pair, including stabilization of the chemical transition state and definition of the 5'-exon/intron boundary.

Animals↗
PubMed1996

Assembly of a ribonucleoprotein catalyst by tertiary structure capture.

CBP2 is an RNA tertiary structure binding protein required for efficient splicing of a yeast mitochondrial group I intron. CBP2 must wait for folding of the two RNA domains that make up the catalytic core before it can bind. In a subsequent step, association of the 5' domain of the RNA is stabilized by additional interactions with the protein. Thus, CBP2 functions primarily to capture otherwise transient RNA tertiary structures. This simple one-RNA, one-protein system has revealed how the kinetic pathway of RNA folding can direct the assembly of a specific ribonucleoprotein complex. There are parallels to steps in the formation of a much more complex ribonucleoprotein, the 30S ribosomal subunit.

Catalysis↗
PubMed1996

Kinetic pathway for folding of the Tetrahymena ribozyme revealed by three UV-inducible crosslinks.

The kinetics of RNA folding were examined in the L-21 ribozyme, an RNA enzyme derived from the self-splicing Tetrahymena intron. Three UV-inducible crosslinks were mapped, characterized, and used as indicators for the folded state of the ribozyme. Together these data suggest that final structures are adopted first by the P4-P6 independently folding domain and only later in a region that positions the P1 helix (including the 5' splice site), a region whose folding is linked to that of a portion of the catalytic core. At intermediate times, a non-native structure forms in the region of the triple helical scaffold, which connects the major folding domains. At 30 degrees C, the unfolded ribozyme passes through these stages with a half-life of 2 min from the time magnesium cations are provided. At higher temperatures, the half-life is shortened but the order of events is unchanged. Thermal melting of the fully folded ribozyme also revealed a multi-stage process in which the steps of folding are reversed: the kinetically slowest structure is the least stable and melts first. These structures of the ribozyme also bind Mg2+ cooperatively and their relative affinity for binding seems to be a major determinant in the order of events during folding. Na+ can also substitute for Mg2+ to give rise to the same crosslinkable structures, but only at much higher concentrations. Specific binding sites for Mg2+ may make this cation particularly efficient at electrostatic stabilization during folding of these ribozyme structures.

Animals↗
PubMed1996

X-ray crystallography of large RNAs: heavy-atom derivatives by RNA engineering.

For small RNAs, isomorphous heavy-atom derivatives can be obtained by crystallizing synthetic versions that incorporate modified nucleotides such as iodo- or bromouridine. However, such a synthetic approach is not yet feasible for RNAs greater than approximately 40 nt. We have been investigating P4-P6, a 160-nt domain of the self-splicing Tetrahymena intron whose structure was solved recently (Cate JH et al., 1996, Science 273:1678-1685). To incorporate iodouridine, a two-piece RNA was constructed. The 5' segment, containing the majority of the molecule, was transcribed in vitro using a self-processing hammerhead ribozyme to cleave the nascent transcript and give a homogenous 3' end. A synthetic 5-iodouridine-containing RNA corresponding to the remainder of the sequence was then annealed to the transcribed piece of RNA. The resulting RNA appeared structurally and functionally sound as judged by nondenaturing gel electrophoresis and RNA cleavage assays. Four versions of this two-piece system with 5-iodouridine substitutions at different positions crystallized under the same conditions as the native RNA, yielding two useful heavy-atom derivatives of P4-P6. The position of the iodine atoms for the derivatives could be determined in the absence of phase information, and an interpretable electron density map was calculated using only the data from the two iodouridine derivatives. This approach is expected to be readily adaptable to other large, structured RNA molecules.

Animals↗