Tetrahymena Thermophila Ribozyme Research Articles

A split ribozyme that links detection of a native RNA to orthogonal protein outputs

Individual RNA remains a challenging signal to synthetically transduce into different types of cellular information. Here, we describe Ribozyme-ENabled Detection of RNA (RENDR), a plug-and-play strategy that uses cellular transcripts to template the assembly of split ribozymes, triggering splicing reactions that generate orthogonal protein outputs. To identify split ribozymes that require templating for splicing, we use laboratory evolution to evaluate the activities of different split variants of the Tetrahymena thermophila ribozyme. The best design delivers a 93-fold dynamic range of splicing with RENDR controlling fluorescent protein production in response to an RNA input. We further resolve a thermodynamic model to guide RENDR design, show how input signals can be transduced into diverse outputs, demonstrate portability across different bacteria, and use RENDR to detect antibiotic-resistant bacteria. This work shows how transcriptional signals can be monitored in situ and converted into different types of biochemical information using RNA synthetic biology.

How to Kinetically Dissect an RNA Machine.

RNA-based machines are ubiquitous in Nature and increasingly important for medicines. They fold into complex, dynamic structures that process information and catalyze reactions, including reactions that generate new RNAs and proteins across biology. What are the experimental strategies and steps that are necessary to understand how these complex machines work? Two 1990 papers from Herschlag and Cech on “Catalysis of RNA Cleavage by the Tetrahymena thermophila Ribozyme” provide a master class in dissecting an RNA machine through kinetics approaches. By showing how to propose a kinetic framework, fill in the numbers, do cross-checks, and make comparisons across mutants and different RNA systems, the papers illustrate how to take a mechanistic approach and distill the results into general insights that are difficult to attain through other means.

In vivo selection of mRNA repair ribozymes

Group I introns are catalytic RNAs (ribozymes) that can excise themselves from pre‐mRNAs. These ribozymes can be taken out of their pre‐mRNA context and modified to replace the 3’‐terminus of a substrate mRNA with their own 3’‐terminus. To employ these trans‐splicing ribozymes for therapeutic applications, it is necessary to improve the in vivo mRNA repair efficiency. We developed an in vivo selection method that is able to increase the mRNA repair efficiency of the trans‐splicing Tetrahymena thermophila ribozyme by selecting from pools with several million ribozyme variants. These ribozyme variants contained 10 or 20 randomized nucleotides in their external guide sequences (EGSs), a region of the ribozyme known to increase splicing efficiency and fidelity. Ribozymes with beneficial EGSs were able to repair a mutated mRNA encoding the chloramphenicol acetyl transferase, and allow growth of the E. coli on medium containing chloramphenicol. Our selections isolated several efficient mRNA repair ribozymes. The predicted secondary structures of the selected EGSs contain several distinct structural motifs. Three of these motifs were previously known, and two motifs are new. Further in vivo selections could allow the generation of ribozymes that repair mRNA efficiently enough for therapeutic applications.

Equilibrium Properties and Force-Driven Unfolding Pathways of RNA Molecules

The mechanical unfolding of a simple RNA hairpin and of a 236-base portion of the Tetrahymena thermophila ribozyme is studied by means of an Ising-like model. Phase diagrams and free energy landscapes are computed exactly and suggest a simple two-state behavior for the hairpin and the presence of intermediate states for the ribozyme. Nonequilibrium simulations give the possible unfolding pathways for the ribozyme, and the dominant pathway corresponds to the experimentally observed one.

Hinge Stiffness Is a Barrier to RNA Folding

Cation-mediated RNA folding from extended to compact, biologically active conformations relies on a temporal balance of forces. The Mg 2 + -mediated folding of the Tetrahymena thermophila ribozyme is characterized by rapid nonspecific collapse followed by tertiary-contact-induced compaction. This article focuses on an autonomously folding portion of the Tetrahymena ribozyme, its P4–P6 domain, in order to probe one facet of the rapid collapse: chain flexibility. The time evolution of P4–P6 folding was followed by global and local measures as a function of Mg 2 + concentration. While all concentrations of Mg 2 + studied are sufficient to screen the charge on the helices, the rates of compaction and tertiary contact formation diverge as the concentration of Mg 2 + increases; collapse is greatly accelerated by Mg 2 + , while tertiary contact formation is not. These studies highlight the importance of chain stiffness to RNA folding; at 10 mM Mg 2 + , a stiff hinge limits the rate of P4–P6 folding. At higher magnesium concentrations, the rate-limiting step shifts from hinge bending to tertiary contact formation.

Concordant Exploration of the Kinetics of RNA Folding from Global and Local Perspectives

Time-resolved small-angle X-ray scattering (SAXS) with millisecond time-resolution reveals two discrete phases of global compaction upon Mg2+-mediated folding of the Tetrahymena thermophila ribozyme. Electrostatic relaxation of the RNA occurs rapidly and dominates the first phase of compaction during which the observed radius of gyration (R(g)) decreases from 75 angstroms to 55 angstroms. A further decrease in R(g) to 45 angstroms occurs in a well-defined second phase. An analysis of mutant ribozymes shows that the latter phase depends upon the formation of long-range tertiary contacts within the P4-P6 domain of the ribozyme; disruption of the three remaining long-range contacts linking the peripheral helices has no effect on the 55-45 angstroms compaction transition. A better understanding of the role of specific tertiary contacts in compaction was obtained by concordant time-resolved hydroxyl radical (OH) analyses that report local changes in the solvent accessibility of the RNA backbone. Comparison of the global and local measures of folding shows that formation of a subset of native tertiary contacts (i.e. those defining the ribozyme core) can occur within a highly compact ensemble whose R(g) is close to that of the fully folded ribozyme. Analyses of additional ribozyme mutants and reaction conditions establish the generality of the rapid formation of a partially collapsed state with little to no detectable tertiary structure. These studies directly link global RNA compaction with formation of tertiary structure as the molecule acquires its biologically active structure, and underscore the strong dependence on salt of both local and global measures of folding kinetics.

Perturbation of the Hierarchical Folding of a Large RNA by the Destabilization of its Scaffold's Tertiary Structure

The P4-P6 domain serves as a scaffold against which the periphery and catalytic core organize and fold during Mg2+-mediated folding of the Tetrahymena thermophila ribozyme. The most prominent structural motif of the P4-P6 domain is the tetraloop-tetraloop receptor interaction which "clamps" the distal parts of its hairpin-like structure. Destabilization of the tertiary structure of the P4-P6 domain by perturbation of the tetraloop-tetraloop receptor interaction alters the Mg2+-mediated folding pathway. The folding hierarchy of P5c approximately P4-P6 > periphery > catalytic core that is a striking attribute of the folding of the wild-type RNA is abolished. The initial steps in folding of the mutant RNA are > or =50-fold faster than those of the wild-type ribozyme with the earliest observed tertiary contacts forming around regions known to specifically bind Mg2+. The interaction between the mutant tetraloop and the tetraloop receptor appears coincidently with slowly forming catalytic core tertiary contacts. Thus, the stability conferred upon the P4-P6 domain by the tetraloop-tetraloop receptor interaction dictates the preferred folding pathway by stabilizing an early intermediate. A sub-denaturing concentration of urea diminishes the early barrier to folding the wild-type ribozyme along with complex effects on the subsequent steps of folding the wild-type and mutant RNA.

Monovalent Ion-mediated Folding of the Tetrahymena thermophila Ribozyme

The time-course of monovalent cation-induced folding of the L-21 Sca1 Tetrahymena thermophila ribozyme and a selected mutant was quantitatively followed using synchrotron X-ray (.OH) footprinting. Initiating folding by increasing the concentration of either Na+ or K+ to 1.5M from an initial condition of approximately 0.008 M Na+ at 42 degrees C resulted in the complete formation of tertiary contacts within the P5abc subdomain and between the peripheral helices within the dead time of our measurements (k>50 s(-1)). These results contrast with folding rates of 2-0.2 s(-1) previously observed for formation of these contacts in 10mM Mg2+ from the same initial condition. Thus, the initial formation of native tertiary contacts is inhibited by divalent but not monovalent cations. The native contacts within the catalytic core form without a detectable burst phase at rates of 0.4-1.0 s(-1) in a manner reminiscent of the Mg2+-dependent folding behavior, although tenfold faster. The tertiary interactions stabilizing the catalytic core interaction with P4-P6 and P2.1, as well as one of the protections internal for the P4-P6 domain, display progress curves with appreciable burst amplitudes and a phase comparable in rate to that of the catalytic core. That the slow folding of the ribozyme's core is a consequence of the alt-P3 secondary structure is shown by the 100% burst phase amplitudes that are observed for folding of the U273A mutant ribozyme within which the native secondary structure (P3) is strengthened. Thus, formation of a misfolded intermediate(s) resulting from the alt-P3 secondary structure is independent of ion valency while the rate at which the respective intermediates are resolved is sensitive to ion valency. The overall portrait painted by these results is that ion valency differentially affects steps in the folding process and that folding in monovalent ion alone for the U273A mutant Tetrahymena ribozyme is fast and direct.

Multiple Monovalent Ion-dependent Pathways for the Folding of the L-21 Tetrahymena thermophila Ribozyme

Synchrotron hydroxyl radical (·OH) footprinting is a technique that monitors the local changes in solvent accessibility of the RNA backbone on milliseconds to minutes time-scales. The Mg 2+-dependent folding of the L-21 Sca 1 Tetrahymena thermophila ribozyme has been followed using this technique at an elevated concentration of monovalent ion (200 mM NaCl) and as a function of the initial annealing conditions and substrate. Previous studies conducted at low concentrations of monovalent ion displayed sequential folding of the P4–P6 domain, the peripheral helices and the catalytic core, with each protection displaying monophasic kinetics. For ribozyme annealed in buffer containing 200 mM NaCl and folded by the addition of 10 mM MgCl 2, multiple kinetic phases are observed for ·OH protections throughout the ribozyme. The independently folding P4–P6 domain is the first to fold with its protections displaying 50–90% burst phase amplitudes. That the folding of P4–P6 within the ribozyme does not display the 100% burst phase of isolated P4–P6 at 200 mM NaCl shows that interactions with the remainder of the ribozyme impede this domain's folding. In addition, ·OH protections constituting each side of a tertiary contact are not coincident in some cases, consistent with the formation of transient non-native interactions. While the peripheral contacts and triple helical scaffold exhibit substantial burst phases, the slowest protection to appear is J8/7 in the catalytic core, which displays a minimal burst amplitude and whose formation is coincident with the recovery of catalytic activity. The number of kinetic phases as well as their amplitudes and rates are different when the ribozyme is annealed in low-salt buffer and folded by the concomitant addition of monovalent and divalent cations. Annealed substrate changes the partitioning of the ribozyme among the multiple folding populations. These results provide a map of the early steps in the ribozyme's folding landscape and the degree to which the preferred pathways are dependent upon the initial reaction conditions.

Monovalent cations mediate formation of native tertiary structure of the Tetrahymena thermophila ribozyme.

The formation of individual tertiary contacts of the Tetrahymena L-21 Sca I ribozyme has been monitored by hydroxyl radical footprinting and its global conformation by analytical ultracentrifugation as a function of monovalent ion concentration in the absence of divalent ions. Advanced methods of data analysis, which allow the hydroxyl radical reactivity of every nucleotide to be quantified, permit monitoring of each and every structural element of the RNA. Monovalent ion-mediated global compaction of the ribozyme is accompanied by the formation of native tertiary contacts; most native tertiary contacts are evident except several that are located near where divalent ions are observed in crystallographic structures. Non-native tertiary contacts are also observed at low but not high concentrations of monovalent ions. In light of recent studies that have shown that the presence of monovalent ions greatly accelerates the Mg2+-dependent folding of the Tetrahymena ribozyme, the present studies suggest that Na+ concentration changes not only the starting position of the RNA on its folding funnel but also pushes it deep into the well by forming native tertiary contacts and, thus, favoring fast and correct folding pathways.

Playing with RNA molecules

Scientists at the University of California (Berkley, CA, USA) have developed a technique to study the folding and unfolding of single RNA molecules using mechanical force. Carlos Bustamante and colleagues attached each end of three different types of RNA molecules: a simple RNA hairpin, an RNA molecule containing a three-helix junction, and the P5abc domain of the Tetrahymena thermophila ribozyme, to a microscopic plastic bead [Science (2001) 292, 733–737]. A piezoelectric actuator was then used to supply the force needed to unfold the molecule and the intensity of this force was measured by a laser beam. The rates of folding and unfolding of the single RNA molecules, and the positions of their transitions states along the reaction coordinate, were thus determined. AR(http://www.hhmi.org/news/bustamante.html).

New loop-loop tertiary interactions in self-splicing introns of subgroup IC and ID: a complete 3D model of the Tetrahymena thermophila ribozyme.

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 non-conserved 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. We present phylogenetic and experimental evidence for the existence of three additional tertiary base pairings between hairpin loops within peripheral components of subgroup IC1 and ID introns. Each of these new long range interactions, called P13, P14 and P16, involves a terminal loop located in domain 2. Although domains 2 of IC and ID introns share very strong sequence similarity, their terminal loops interact with domains 5 and 9 (subgroup IC1) and domain 6 (subgroup ID). Based on these tertiary contacts, comparative sequence analysis, and published experimental results such as Fe(II)-EDTA protection patterns, we propose 3D models for two entire group I introns, the subgroup IC1 intron in the large ribosomal precursor RNA of Tetrahymena thermophila and the SdCob.1 subgroup ID intron found in the cytochrome b gene of Saccharomyces douglasii. Three-dimensional models of group I introns belonging to four different subgroups are now available. They all emphasize the modular and hierarchical organization of the architecture of group I introns and the widespread use of base-pairings between terminal hairpin loops for stabilizing the folded and active structures of large and complex RNA molecules.

P-B3-03 X-ray “footprinting” analysis of the Mg 2+ dependent folding of the Tetrahymena thermophila ribozyme : Sclavi † B, Woodson § S, Sullivan † M, Chance †‡ M, Brenowitz ‡ M. Depts. of Physiology and Biophysics † and Biochemistry ‡, Albert Einstein College of Medicine, Bronx, NY, (USA) and Dept. of Biochemistry §, Univ. of Maryland, College Park, MD, (USA)

P-B3-03 X-ray “footprinting” analysis of the Mg 2+ dependent folding of the Tetrahymena thermophila ribozyme : Sclavi † B, Woodson § S, Sullivan † M, Chance †‡ M, Brenowitz ‡ M. Depts. of Physiology and Biophysics † and Biochemistry ‡, Albert Einstein College of Medicine, Bronx, NY, (USA) and Dept. of Biochemistry §, Univ. of Maryland, College Park, MD, (USA)

Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 2. Kinetic description of the reaction of an RNA substrate that forms a mismatch at the active site

The site-specific endonuclease reaction catalyzed by the ribozyme from the Tetrahymena pre-rRNA intervening sequence has been characterized with a substrate that forms a "matched" duplex with the 5' exon binding site of the ribozyme [G2CCCUCUA5 + G in equilibrium with G2CCCUCU + GA5 (G = guanosine); Herschlag, D., & Cech, T.R. (1990) Biochemistry (preceding paper in this issue)]. The rate-limiting step with saturating substrate is dissociation of the product G2CCCUCU. Here we show that the reaction of the substrate G2CCCGCUA5, which forms a "mismatched" duplex with the 5' exon binding site at position -3 from the cleavage site, has a value of kcat that is approximately 10(2)-fold greater than kcat for the matched substrate (50 degrees C, 10 mM MgCl2, pH 7). This is explained by the faster dissociation of the mismatched product, G2CCCGCU, than the matched product. With subsaturating oligonucleotide substrate and saturating G, the binding of the oligonucleotide substrate and the chemical step are each partially rate-limiting. The rate constant for the chemical step of the endonuclease reaction and the rate constant for the site-specific hydrolysis reaction, in which solvent replaces G, are each within approximately 2-fold with the matched and mismatched substrates, despite the approximately 10(3)-fold weaker binding of the mismatched substrate. This can be described as "uniform binding" of the base at position -3 in the ground state and transition state [Albery, W.J., & Knowles, J. R. (1976) Biochemistry 15, 5631-5640]. Thus, the matched substrate does not use its extra binding energy to preferentially stabilize the transition state.(ABSTRACT TRUNCATED AT 250 WORDS)

Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 1. Kinetic description of the reaction of an RNA substrate complementary to the active site

A ribozyme derived from the intervening sequence (IVS) of the Tetrahymena preribosomal RNA catalyzes a site-specific endonuclease reaction: G2CCCUCUA5 + G in equilibrium with G2CCCUCU + GA5 (G = guanosine). This reaction is analogous to the first step in self-splicing of the pre-rRNA, with the product G2CCCUCU analogous to the 5'-exon. The following mechanistic conclusions have been derived from pre-steady-state and steady-state kinetic measurements at 50 degrees C and neutral pH in the presence of 10 mM Mg2+. The value of kcat/Km = 9 x 10(7) M-1 min-1 for the oligonucleotide substrate with saturating G represents rate-limiting binding. This rate constant for binding is of the order expected for formation of a RNA.RNA duplex between oligonucleotides. (Phylogenetic and mutational analyses have shown that this substrate is recognized by base pairing to a complementary sequence within the IVS). The value of kcat = 0.1 min-1 represents rate-limiting dissociation of the 5'-exon analogue, G2CCCUCU. The product GA5 dissociates first from the ribozyme because of this slow off-rate for G2CCCUCU. The similar binding of the product, G2CCCUCU, and the substrate, G2CCCUCUA5, to the 5'-exon binding site of the ribozyme, with Kd = 1-2 nM, shows that the pA5 portion of the substrate makes no net contribution to binding. Both the substrate and product bind approximately 10(4)-fold (6 kcal/mol) stronger than expected from base pairing with the 5'-exon binding site. Thus, tertiary interactions are involved in binding. Binding of G2CCCUCU and binding of G are independent. These and other data suggest that binding of the oligonucleotide substrate, G2CCCUCUA5, and binding of G are essentially random and independent. The rate constant for reaction of the ternary complex is calculated to be kc approximately equal to 350 min-1, a rate constant that is not reflected in the steady-state rate parameters with saturating G. The simplest interpretation is adopted, in which kc represents the rate of the chemical step. A site-specific endonuclease reaction catalyzed by the Tetrahymena ribozyme in the absence of G was observed; the rate of the chemical step with solvent replacing guanosine, kc(-G) = 0.7 min-1, is approximately 500-fold slower than that with saturating guanosine. The value of kcat/Km = 6 x 10(7) M-1 min-1 for this hydrolysis reaction is only slightly smaller than that with saturating guanosine, because the binding of the oligonucleotide substrate is predominantly rate-limiting in both cases.(ABSTRACT TRUNCATED AT 400 WORDS)