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T R Cech

Publications and source records attributed to T R Cech.

At least 73 records · Page 4Linked to original sources
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↗
PubMed1995

Relative orientation of RNA helices in a group 1 ribozyme determined by helix extension electron microscopy.

The relative orientation of helical elements in a folded RNA molecule provides key information about its three-dimensional architecture. We have developed a method that involves extending peripheral helices of an RNA, mounting for electron microscopy in the absence of protein and measuring interhelical angles. As a control, extended anticodon and acceptor stems of tRNA(Phe) were found to form a 92 +/- 20 degrees angle, consistent with the X-ray structure. Single, double and triple extensions (50-80 bp) of helical elements P2.1, P6b and P8 of the Tetrahymena group I ribozyme did not alter its catalytic activity. The measured angle between P6b and P8 is consistent with the Michel-Westhof structural model, while the P2.1-P6b and P2.1-P8 angles allow P2.1 to be positioned in the model. The angle distributions of the ribozyme are broader than those of the tRNA, which may reflect the dynamics of the RNA. Helix extension allows low-resolution electron microscopy to provide much higher resolution information about the disposition of helical elements in RNA. It should be applicable to diverse RNAs and ribonucleoprotein complexes.

Animals↗
PubMed1995

Protein facilitation of group I intron splicing by assembly of the catalytic core and the 5' splice site domain.

The yeast mitochondrial group I intron b15 undergoes self-splicing at high Mg2+ concentrations, but requires the splicing factor CBP2 for reaction under physiological conditions. Chemical accessibility and UV cross-linking experiments now reveal that self-processing is slow because functional elements are not properly positioned in an active tertiary structure. Folding energy provided by CBP2 drives assembly of two RNA domains that comprise the catalytic core and meditates association of an approximately 100 nt 5' domain that contains the 5' splice site. Thus, the protein assembles RNA secondary structure elements into a specific three-dimensional array while the RNA provides the catalytic center. The division of labor between RNA and protein illustrated by this simple system reveals principles applicable to complex ribonucleoprotein assemblies such as the spliceosome and ribosome.

Base Sequence↗
PubMed1995

Efficient protein-facilitated splicing of the yeast mitochondrial bI5 intron.

The splicing factor CBP2 is required to excise the yeast mitochondrial group I intron bI5 in vivo and at low magnesium ion concentrations in vitro. CBP2 binding is strengthened 20-fold by increasing Mg2+ concentrations from 5 to 40 mM, implying the protein binds, in part, to the same structure as that stabilized by the cation. The same transition is also observed as a cooperative increase in the rate of self-processing between 5 and 40 mM Mg2+, providing strong evidence for an RNA folding transition promoted by either Mg2+ or CBP2. The first step of splicing, guanosine addition at the 5' splice site, is rate limiting for exon ligation. At low (5 mM) magnesium ion, reaction (measured as kcat/Km or kcat) is accelerated 3 orders of magnitude by saturating CBP2. At near-saturating Mg2+ (40 mM), acceleration is 8- and 30-fold, for kcat and kcat/Km, respectively, so high magnesium ion concentrations fail to compensate completely for protein facilitation. Thus, self-splicing proceeds via two additional transitions as compared with reaction of the bI5-CBP2 complex, only the first of which is efficiently promoted by the cation. Guanosine 5'-monophosphate binds (Kd approximately 0.3 mM) with the same affinity to bI5 and the bI5-protein complex, supporting independent binding of the nucleophile and CBP2. Substitution of a phosphorothioate at the 5' splice site and pH profiles provide evidence that kcat is limited by chemistry at low pH and by a conformational step at high pH. Because binding by either Mg2+ or CBP2 increases the rate of chemistry more than the rate of the conformational step, in the physiological pH range (7-7.6) the protein-facilitated reaction is limited by a conformation step while self-splicing reaction is limited by chemistry. We conclude that CBP2 makes manifold contributions to bI5 splicing: binding compensates for at least two structural defects and accelerates the rate of the chemistry.

Base Sequence↗
PubMed1995

Phosphorylation of the Oxytricha telomere protein: possible cell cycle regulation.

In the macronucleus of the ciliate Oxytricha nova, telomeres end with single-stranded (T4G4)2 DNA bound to a heterodimeric telomere protein (alpha beta). Both the alpha and beta subunits (alpha-TP and beta-TP) were phosphorylated in asynchronously growing Oxytricha; beta-TP was phosphorylated to a much higher degree. In vitro, mouse cyclin-dependent kinases (Cdks) phosphorylated beta-TP in a lysine-rich domain that is not required for specific DNA binding but is implicated in higher order structure formation of telomeres. Therefore, phosphorylation of beta-TP could modulate a function of the telomere protein that is separate from specific DNA binding. Phosphoamino acid analysis revealed that the mouse Cdks modify predominantly threonine residues in beta-TP, consistent with the observation that beta-TP contains two consensus Cdk recognition sequences containing threonine residues. In Xenopus egg extracts that undergo cell cycling, beta-TP was phosphorylated in M phase and dephosphorylated in interphase. This work provides the first direct evidence of phosphorylation at telomeres in any organism, as well as indirect evidence for cell cycle regulation of telomere phosphorylation. The Cdc2/cyclin A and Cdc2/cyclin B kinases are required for major mitotic events. An attractive model is that phosphorylation of beta-TP by these kinases is required for the breakdown of telomere associations with each other and/or with nuclear structures prior to nuclear division.

Amino Acid Sequence↗
PubMed1995

A positive entropy change for guanosine binding and for the chemical step in the Tetrahymena ribozyme reaction.

The ribozyme derived from the group I intron of Tetrahymena thermophila binds an exogenous guanosine nucleotide, which acts as the nucleophile in the sequence-specific cleavage of oligonucleotides. By examining the temperature dependence of the reaction under conditions where Km = Kd, we conclude the following: (1) Guanosine 5'-monophosphate (pG) binds to the closed ribozyme-oligonucleotide substrate complex with a positive entropy change (delta S degree' = +23 eu) and an enthalpy change (delta H degree') close to zero. This is contrary to the expectation that binding would cause increased order (negative delta S degree) and be driven by a negative delta H degree. (2) Inosine and 2-aminopurine riboside, each lacking two hydrogen-bonding moieties relative to guanosine, also bind with a positive entropy value and an unfavorable (positive) delta H degree'. From this result, we suggest that the hydrogen-bonding moieties make an enthalpic contribution to guanosine binding overcoming an intrinsic unfavorable delta H. (3) At 0 degree C, there is equally tight binding of pG in the presence and absence of oligonucleotide substrate bound to the ribozyme. Thus, energetic interactions responsible for the thermodynamic coupling between pG and oligonucleotide substrate binding seen at higher temperatures are indirect. (4) The activation barrier of the chemical step is stabilized by a positive delta S++ (+31 to 39 eu). This stabilization is seen in four reactions using substrates with two different leaving groups in the presence and absence of pG, suggesting that the entropic contribution is inherent to the active site. The positive delta S values for the chemical step and for the binding of pG can be explained by a conformational change or release of water. Thus, although hydrogen bonding contributes to binding of nucleotides to this RNA enzyme as previously thought, it is these other events which produce a positive delta S that provide the energetic driving force for binding.

Animals↗