An ultraviolet-inducible adenosine-adenosine cross-link reflects the catalytic structure of the Tetrahymena ribozyme (original) (raw)
Related papers
Biochemistry, 1987
We have converted the intramolecular cyclization reaction of the self-splicing intervening sequence (IVS) ribonucleic acid (RNA) from Tetrahymena thermophila into an intermolecular guanosine addition reaction. This was accomplished by selectively removing the 3'4erminal nucleotide by oxidation and @-elimination; the @-eliminated IVS thereby is no longer capable of reacting with itself. However, under cyclization conditions, a free guanosine molecule can make a nucleophilic attack at the normal cyclization site. We have used this guanosine addition reaction as a model system for a Michaelis-Menten kinetic analysis
A Mechanistic Framework for the Second Step of Splicing Catalyzed by the Tetrahymena Ribozyme †
Biochemistry, 1996
A simple model system is described which mimics the second step of splicing and reverse cyclization reactions of the self-splicing intron from Tetrahymena thermophila. This model is based on the L-21 Sca I catalyzed ligation reaction between exogenously added oligomers: cucu + UCGa y \ z L-21 Sca I cucua + UCG. Steady-state kinetics for the forward and reverse direction were measured at 15°C to find oligonucleotides that exhibit Michaelis-Menten behavior with acceptable K M s. CUCU and UCGA fit both criteria and were chosen for further studies. Steady-state kinetics reveal a lag that appears to be an RNA folding step that is eliminated by preincubation of the ribozyme with 2 mM and higher [Mg 2+ ] and by UCGA. At constant ionic strength, the Mg 2+ dependence of steady-state rates exhibits a sharp maximum near 5 mM Mg 2+ . Pre-steady-state and steady-state kinetics, along with activesite titrations, explain the Mg 2+ profile: the rate of reaction up to and including chemistry increases with Mg 2+ concentration, while the fraction of active ribozyme and the rate of postchemistry steps decrease with Mg 2+ concentration. The rate-limiting step at 5 mM Mg 2+ for the reaction mimicking the second step of splicing is either chemistry or a conformational change before chemistry involving ribozyme bound with substrates. The rate-limiting step at 50 mM Mg 2+ appears to be a postchemistry conformational change of the ribozyme or product release. At 50 mM Mg 2+ , single-turnover experiments support ordered binding of substrates with 5′-exon mimic binding before 3′-splice site mimic. Moreover, the 3′-splice site mimic binds and reacts in the presence of 5′-exon mimics predocked into the catalytic core. Results also indicate that Mg 2+ ions associate with the ribozyme upon docking.
Cell, 1982
In the macronuclear rRNA genes of Tetrahymena thermophila, a 413 bp intervening sequence (IVS) interrupts the 26S rRNA-coding region. A restriction fragment of the rONA containing the IVS and portions of the adjacent rRNA sequences (exons) was inserted downstream from the lac UVS promoter in a recombinant plasmid. Transcription of this template by purified Escherichia coli RNA polymerase in vitro produced a shortened version of the pre-rRNA, which was then deproteinized. When incubated with monovalent and divalent cations and a guanosine factor, this RNA underwent splicing. The reactions that were characterized included the precise excision of the IVS, attachment of guanosine to the 5' end of the IVS, covalent cyclization of the IVS and ligation of the exons. We conclude that splicing activity is intrinsic to the structure of the RNA, and that enzymes, small nuclear RNAs and folding of the pre-rRNA into an RNP are unnecessary for these reactions. We propose that the IVS portion of the RNA has several enzyme-like properties that enable it to break and reform phosphodiester bonds. The finding of autocatalytic rearrangements of RNA molecules has implications for the mechanism and the evolution of other reactions that involve RNA.
Biochemistry, 1996
Self-splicing of Tetrahymena pre-rRNA proceeds in two consecutive phosphoryl transesterification steps. One major difference between these steps is that in the first an exogenous guanosine (G) binds to the active site, while in the second the 3′-terminal G414 residue of the intron binds. The first step has been extensively characterized in studies of the L-21Scal ribozyme, which uses exogenous G as a nucleophile. In this study, mechanistic features involved in the second step are investigated by using the L-21G414 ribozyme. The L-21G414 reaction has been studied in both directions, with G414 acting as a leaving group in the second step and a nucleophile in its reverse. The rate constant of chemical step is the same with exogenous G bound to the L-21ScaI ribozyme and with the intramolecular guanosine residue of the L-21G414 ribozyme. The result supports the previously proposed single G-binding site model and further suggests that the orientation of the bound G and the overall active site structure is the same in both steps of the splicing reaction. An evolutionary rationale for the use of exogenous G in the first step is also presented. The results suggest that the L-21G414 ribozyme exists predominantly with the 3′-terminal G414 docked into the G-binding site. This docking is destabilized by ∼100-fold when G414 is attached to an electron-withdrawing pA group. The internal equilibrium with K int) 0.7 for the ribozyme reaction indicates that bound substrate and product are thermodynamically matched and is consistent with a degree of symmetry within the active site. These observations are consistent with the presence of a second Mg ion in the active site. Finally, the slow dissociation of a 5′ exon analog relative to a ligated exon analog from the L-21G414 ribozyme suggests a kinetic mechanism for ensuring efficient ligation of exons and raises new questions about the overall self-splicing reaction.
The chemical basis of adenosine conservation throughout the Tetrahymena ribozyme
RNA, 1998
Adenosines are present at a disproportionately high frequency within several RNA structural motifs. To explore the importance of individual adenosine functional groups for group I intron activity, we performed Nucleotide Analog Interference Mapping (NAIM) with a collection of adenosine analogues. This paper reports the synthesis, transcriptional incorporation, and the observed interference pattern throughout the Tetrahymena group I intron for eight adenosine derivatives tagged with an a-phosphorothioate linkage for use in NAIM. All of the analogues were accurately incorporated into the transcript as an A. The sites that interfere with the 39-exon ligation reaction of the Tetrahymena intron are coincident with the sites of phylogenetic conservation, yet the interference patterns for each analogue are different. These interference data provide several biochemical constraints that improve our understanding of the Tetrahymena ribozyme structure. For example, the data support an essential A-platform within the J6/6a region, major groove packing of the P3 and P7 helices, minor groove packing of the P3 and J4/5 helices, and an axial model for binding of the guanosine cofactor. The data also identify several essential functional groups within a highly conserved single-stranded region in the core of the intron (J8/7). At four sites in the intron, interference was observed with 29-fluoro A, but not with 29-deoxy A. Based upon comparison with the P4-P6 crystal structure, this may provide a biochemical signature for nucleotide positions where the ribose sugar adopts an essential C29-endo conformation. In other cases where there is interference with 29-deoxy A, the presence or absence of 29-fluoro A interference helps to establish whether the 29-OH acts as a hydrogen bond donor or acceptor. Mapping of the Tetrahymena intron establishes a basis set of information that will allow these reagents to be used with confidence in systems that are less well understood.
Sequence requirements for self-splicing of the Tetrahymena thermophila pre-ribosomal RNA
Nucleic Acids Research, 1985
The sequence requirements for splicing of the Tetrahymena pre-rRKA have been examined by altering the rRNA gene to produce versions that contain insertions and deletions within the intervening sequence (IVS). The altered genes were transcribed and the RNA tested for self-splicing in vitro. A number of insertions (8-54 nucleotides) at three locations had no effect on self-splicing activity. Two of these insertions, located at a aite 5 nucleotldas preceding the 3'-end of the IVS, did not alter the choice of the 3 1 splice site. Thus the 3' splice site is not chosen by its distance from a fixed point within the IVS. Analysis of deletions constructed at two sites revealed two structures, a hairpin loop and a stem-loop, that are entirely dispensable for IVS excision in vitro. Three other regions were found to be necessary. The regions that are important for self-splicing are not restricted to the conserved sequence elements that define this class of intervening sequences. The requirement for structures within the IVS for pre-rRNA splicing is in sharp contrast to the very limited role of IVS structure in nuclear pra-mRNA splicing.
Selection of small molecules by the Tetrahymena catalytic center
Nucleic Acids Research, 1991
The catalytic center in group I RNAs contains a selective binding site that accommodates both guanosine and L-arglnine. In order to understand the specificity of the RNA for small molecules, we analyzed 6 RNAs that vary in this region. Specificity for nucleotldes resides substantially in G264 rather than its paired nucleotlde C311, and is expressed substantially in K^, with comparatively little variation in kcat. ltd is not notably perturbed even for RNAs with mispairs In the active-site helix. For 5 of 6 sequences, effects of RNA substitutions on arginine binding and GTP reactivity are proportional, confirming that arginine contacts a subset of the groups occupied by G. As a result of particular mutations, reaction with GTP is decreased, and reaction with the natural nucleotldes UTP and ATP Is enhanced. Molecular modeling of these effects suggests that exceptionally flexible placement of reactants may be an essential quality of RNAcatalyzed splicing. The specificity of the intron can be rationalized by a type of binding model not previously considered, in which the G/arginine site includes adjacent nucleotides (an 'axial' site), rather than a single nucleotide, G264.
Chemistry & Biology, 1996
Group I introns self-splice via two consecutive trans-esterification reactions in the presence of guanosine cofactor and magnesium ions. Comparative sequence analysis has established that a catalytic core of about 120 nucleotides is conserved in all known group I introns. This core is generally not sufficient for activity, however, and most self-splicing group I introns require nonconserved peripheral elements to stabilize the complete three-dimensional (3D) structure. The physico-chemical properties of group I introns make them excellent systems for unraveling the structural basis of the RNA-RNA interactions responsible for promoting the self-assembly of complex RNAs.
Nucleic Acids Research, 1989
The self-splicing intervening sequence from the precursor rRNA of Tetrahymena thermophila cyclizes to form a covalently closed circle. This circle can be reopened by reaction with oligonucleotide~+or water. The kinetics of circle opening as a function of substrate and Mg concentrations have been measured for dCrU, rCdU, dCdT, and H20 addition. Comparisons with previous results for rCrU suggest: (1) the 2' OH of the 5' sugar of a dinucleoside phosphate is involved in substrate binj4ng, and (2) the 2' OH of the 3' sugar of a dimer substrWe is involved in Mg binding. Evidently, the binding site for a required Mg ion isdependent on both the ribozyme and the dimer substrate. The apparent activation energy and entropy for circle opening by hydrolysis are 31 kcal/mol and 50 eu, respectively. The large, positive activation entropy suggests a partial unfolding of the ribozyme is required for reaction.
Biochemistry, 1994
There is a phylogenetically conserved G U pair at the 5'-splice site of group I introns. When this is mutagenized to a G-C pair, splicing of these introns is greatly reduced. We have used a ribozyme derived from the Tetrahymena group I intron to compare the binding and reactivity of oligonucleotides that form either a G U or a G-C pair at this position. Ribozyme binding of oligonucleotides at 42 "C was measured by native gel electrophoresis and equilibrium dialysis. Binding of GGCCCUCC (C(-1)P), which base-pairs with the ribozyme guide sequence to form a G-C at the cleavage site, was 10-fold weaker than the binding of GGCCCUCU (U(-l)P), which maintains the conserved G U pair at the cleavage site. This is surprising since a terminal G-C enhances the binding between oligonucleotides by 20-fold relative to a terminal G U . Thermal denaturation studies indicate that C(-l)P and several analogs with deoxy substitutions bind the guide-sequence oligonucleotide, GGAGGGAAA, as strongly as they bind the ribozyme. In contrast, U(-l)P binds 240-fold more strongly to the ribozyme than to GGAGGGAAA, a difference that is decreased by deoxy substitutions. Thus, while U(-l)P binds the ribozyme through a combination of base-pairing and specific 2-OH and other tertiary interactions, C(-l)P may bind by basepairing alone. The substrate GGCCCUCCAAAAA (C(-1)s) is cleaved 100-fold more slowly than GGCCCUCUAAAAA (U(-1)s) and also has a higher propensity to be cleaved at the wrong nucleotide position. Taken together, the results suggest that a G-C pair at the ribozyme cleavage site makes docking of the guide-sequencewbstrate helix into the catalytic site less favorable than a G U pair. The resulting consequences of weaker binding, slower reaction, and reduced cleavage fidelity provide a rationale for the phylogenetic conservation of the G U .