Structural basis for Diels-Alder ribozyme-catalyzed carbon-carbon bond formation

The majority of structural efforts addressing RNA's catalytic function have focused on natural ribozymes, which catalyze phosphodiester transfer reactions. By contrast, little is known about how RNA catalyzes other types of chemical reactions. We report here the crystal structures of a ribozyme that catalyzes enantioselective carbon-carbon bond formation by the Diels-Alder reaction in the unbound state and in complex with a reaction product. The RNA adopts a lambda-shaped nested pseudoknot architecture whose preformed hydrophobic pocket is precisely complementary in shape to the reaction product. RNA folding and product binding are dictated by extensive stacking and hydrogen bonding, whereas stereoselection is governed by the shape of the catalytic pocket. Catalysis is apparently achieved by a combination of proximity, complementarity and electronic effects. We observe structural parallels in the independently evolved catalytic pocket architectures for ribozyme- and antibody-catalyzed Diels-Alder carbon-carbon bond-forming reactions.     

The enzyme activity allosteric regulation model based on the composite nature of catalytic and regulatory sites concept

A new kinetic model of enzymatic catalysis is proposed, which postulates that enzyme solutions are equilibrium systems of oligomers differing in the number of subunits and in the mode of their assembly. It is suggested that the catalytic and regulatory sites of allosteric enzymes are of composite nature and appear as a result of subunits joining. Two possible joining modes are postulated at each oligomerization step. Catalytic site may arise on oligomer formed only by one of these modes. Effector acts by fastening together components of certain oligomeric form and increases the life time of this form. It leads to a shift of oligomer equilibrium and increases a proportion of effector-binding oligomers. Effectors-activators bind the oligomers carrying composite catalytic sites and effectors-inhibitors bind the oligomers, which do not carry active catalytic sites. Thus, catalytic activity control in such system is explained by effector-induced changes of a catalytic sites number, but not of a catalytic site activity caused by changes of subunit's tertiary structure. The postulates of the model do not contradict available experimental data and lead to a new type of general rate equation, which allows to describe and understand the specific kinetic behavior of allosteric enzymes as well as Michaelis type enzymes. All known rate equations of allosteric The equation was tested by modeling the kinetics of human erythrocyte phosphofructokinase. It enabled to reproduce quantitatively the 66 kinetic curves experimentally obtained for this enzyme under different reaction conditions.     

The Enzyme Activity Allosteric Regulation Model Based on the Composite Nature of Catalytic and Regulatory Sites Concept

Abstract

A new kinetic model of enzymatic catalysis is proposed, which postulates that enzyme solutions are equilibrium systems of oligomers differing in the number of subunits and in the mode of their assembly. It is suggested that the catalytic and regulatory sites of allosteric enzymes are of composite nature and appear as a result of subunits joining. Two possible joining modes are postulated at each oligomerization step. Catalytic site may arise on oligomer formed only by one of these modes. Effector acts by fastening together components of certain oligomeric form and increases the life time of this form. It leads to a shift of oligomer equilibrium and increases a proportion of effector-binding oligomers. Effectors-activators bind the oligomers carrying composite catalytic sites and effectors-inhibitors bind the oligomers, which do not carry active catalytic sites. Thus, catalytic activity control in such system is explained by effector-induced changes of a catalytic sites number, but not of a catalytic site activity caused by changes of subunit's tertiary structure.

The postulates of the model do not contradict available experimental data and lead to a new type of general rate equation, which allows to describe and understand the specific kinetic behavior of allosteric enzymes as well as Michaelis type enzymes. All known rate equations of allosteric

The equation was tested by modeling the kinetics of human erythrocyte phosphofructokinase. It enabled to reproduce quantitatively the 66 kinetic curves experimentally obtained for this enzyme under different reaction conditions.     

Enhancement of ribozyme catalytic activity by a contiguous oligodeoxynucleotide (facilitator) and by 2''-O-methylation.

RNA catalysts (ribozymes) designed to cleave sequences unique to viral RNA's might be developed as therapeutics. For this purpose, they would require high catalytic efficiency and resistance to nucleases. Reported here are two approaches that can be used in combination to improve these properties. First, catalytic efficiency can be improved by oligonucleotides (facilitators) that bind to the substrate contiguously with the 3'-end of the ribozyme. Second, 2'-O-methylation of flanking sequences of the ribozyme increases catalytic activity as well as resistance to nucleases. In combination with a facilitator oligodeoxynucleotide, the cleavage rate was increased 20 fold over that of the unmodified ribozyme.     

Toward the Selection of Ribozymes for 1,3-Dipolar Cycloaddition Reactions

In vitro selection from combinatorial RNA libraries has repeatedly been used to study the catalytic and binding potential of nucleic acids. These selections not only led to RNA sequences catalyzing transformations known from metabolic pathways but also generated novel ribozymes for typical organic reactions. We were interested in 1,3-dipolar cycloaddition reactions, which are important tools for the formation of heterocyclic systems in organic chemistry and might also be found in the hypothetic RNA world. Here we describe our strategy and experiments to isolate RNA molecules catalyzing a 1,3-dipolar cycloaddition between nitrile oxides and an acrylate conjugated to RNA. We used direct selection with linker-coupled reactants, which has previously allowed the generation of true trans-acting catalysts for bimolecular reactions. A photocleavable linker was introduced to provide for a more stringent selection criterion. The 1,3-dipolar cycloaddition reaction was established in aqueous solution using a modified dinucleotide that was tethered to the dipolarophilic substrate. Two selection protocols were established, namely, a low-stringency affinity-based selection protocol, and a high-stringency procedure using the photocleavable moiety. In neither case was an increased activity toward the desired reaction obtained after 15 and 11 selection rounds, respectively. The resulting pools of RNA from several rounds were investigated both in cis and in trans. The limitations of this selection methodology are discussed in comparison with other catalysts for dipolar cycloadditions and, also, with respect to the unconventional substrates used.     

Allosteric hammerhead ribozyme TRAPs

A new mode of allosteric regulation of nucleic acid enzymes is described and shown to operate effectively with hammerhead ribozymes. In the "TRAP" design (for targeted ribozyme-attenuated probe), a 3' terminal "attenuator" anneals to conserved bases in the catalytic core to form the "off" state of the ribozyme. Binding of RNA or DNA to an antisense sequence linking the ribozyme and attenuator frees the core to fold into an active conformation, even though the antisense sequence itself does not interfere with the ribozyme. TRAP hammerheads based on the previously characterized HH8 ribozyme were shown to be activated more than 250-fold upon addition of the sense strand. RNA oligonucleotides were more effective activators than DNA oligos, consistent with the known relative helix stabilities (RNA-RNA > RNA-DNA). Oligonucleotides that directly paired with the attenuator gave up to 1760-fold activation. The magnitude of the activation was greater when the oligo was added prior to folding than if it was added during the cleavage reaction. The TRAP design requires no prior knowledge of (deoxy)ribozyme structure beyond identification of the essential core. Thus, this approach should be readily generalizable to other systems for biomedicine, sensor technology, and additional applications.     

The encoded primary sequence of a rice seed ADP-glucose pyrophosphorylase subunit and its homology to the bacterial enzyme

Rice seed ADP-glucose pyrophosphorylase cDNA clones were isolated by screening a lambda expression library prepared from rice endosperm poly(A+) RNA with a heterologous antibody raised against the spinach leaf enzyme and subsequently by nucleic acid hybridization. One cDNA plasmid, possessing about 1650 nucleotides, was shown by both DNA and RNA sequence analysis to contain the complete ADP-glucose pyrophosphorylase coding sequence of 483 amino acids. The primary sequence displayed a putative leader peptide presumably required for transport of this nuclear encoded protein into the amyloplasts, a differentiated starch containing plastid. The leader peptide, however, showed little sequence homology with transit peptides displayed by other known nuclear encoded proteins localized in the chloroplasts. A comparison of the primary sequence of the putative mature subunit to the Escherichia coli pyrophosphorylase showed two regions displaying significant homology. These two conserved regions contain residues shown previously to be essential for the allosteric regulation and catalytic activity of the E. coli enzyme. Differences in the primary sequences of the plant and bacterial enzyme may reflect the distinct nature of the allosteric effectors that control these enzymes.     

Oligonucleotide facilitators enhance the catalytic activity of RNA-cleaving DNA enzymes.

From in vitro selection studies, DNA structures have been found that cleave target RNA sequence specifically and show a certain similarity to the well-investigated hammerhead ribozymes. Such DNA enzymes are more resistant to nuclease-mediated degradation than RNA enzymes. On the other hand, their cleavage activity is lower than the activity of hammerhead ribozymes. In the present study, we improved the activity of DNA enzymes by adding oligonucleotide facilitators complementary to the 5' and the 3' ends of the substrate to the cleavage reaction. DNA enzyme activity in vitro was monitored under multiple turnover conditions using short RNA model substrates. We have shown that oligonucleotide facilitators strongly enhance the multiple turnover activity of the DNA enzyme reaction. In one of our model systems with a suitable facilitator combination, we were able to observe a more than 200-fold enhancement of the k(cat)/Km value. The comparison of two DNA enzyme-substrate systems showed that the principal effects of the facilitators were independent of the substrate sequence. However, the degree of facilitator effect was noticeably dependent on the basic catalytic efficiency of DNA enzymes. Furthermore, the efficiency of the DNA enzyme reaction with facilitator was compared with the reaction of a DNA enzyme with a stem sequence extended by the sequence of the facilitator. The multiple turnover activity of such a "long DNA enzyme" is higher than the activity of the short DNA enzyme without facilitators. However, when compared with the multiple turnover reactions of the short DNA enzyme with facilitator, the reaction with the long DNA enzyme is considerably slower. The results obtained with our model systems demonstrate that oligonucleotide facilitators enable DNA enzymes to act as effective multiple turnover catalysts by cleavage of RNA substrates.     

QSAR study of the enantiomeric excess in asymmetric catalytic reactions with topological indices and an artificial neural network

The relationships between the enantiomer excess of product in catalytic asymmetric reactions and the structures of the catalysts or reagents in several asymmetric reactions were studied using a backpropagation (BP) neural network with topological indices and their chiral expansions. The trained network can be used to screen new asymmetric catalysts, estimate catalytic effects, design reaction environments, and prove or improve the proposed reaction mechanism.     

RNA-dependent activation of primer RNA production by influenza virus polymerase: different regions of the same protein subunit constitute the two required RNA-binding sites.

The capped RNA primers required for the initiation of influenza virus mRNA synthesis are produced by the viral polymerase itself, which consists of three proteins PB1, PB2 and PA. Production of primers is activated only when the 5'- and 3'-terminal sequences of virion RNA (vRNA) bind sequentially to the polymerase, indicating that vRNA molecules function not only as templates for mRNA synthesis but also as essential cofactors which activate catalytic functions. Using thio U-substituted RNA and UV crosslinking, we demonstrate that the 5' and 3' sequences of vRNA bind to different amino acid sequences in the same protein subunit, the PB1 protein. Mutagenesis experiments proved that these two amino acid sequences constitute the functional RNA-binding sites. The 5' sequence of vRNA binds to an amino acid sequence centered around two arginine residues at positions 571 and 572, causing an allosteric alteration which activates two new functions of the polymerase complex. In addition to the PB2 protein subunit acquiring the ability to bind 5'-capped ends of RNAs, the PB1 protein itself acquires the ability to bind the 3' sequence of vRNA, via a ribonucleoprotein 1 (RNP1)-like motif, amino acids 249-256, which contains two phenylalanine residues required for binding. Binding to this site induces a second allosteric alteration which results in the activation of the endonuclease that produces the capped RNA primers needed for mRNA synthesis. Hence, the PB1 protein plays a central role in the catalytic activity of the viral polymerase, not only in the catalysis of RNA-chain elongation but also in the activation of the enzyme activities that produce capped RNA primers.     

Allosteric regulation of a ribozyme activity through ligand-induced conformational change.

An allosteric ribozyme has been designed using the hammerhead ribozyme as the active site and aflavin-specific RNA aptamer as a regulatory site. We constructed six variants with a series of base pairs in the linker region (stem II). Under single turnover conditions, kinetic studies were carried out in the absence and presence of flavin mononucleotide (FMN). Interestingly, FMN addition did not influence the cleavage rate of constructs with a 5-6 bp linker but stimulated the catalytic activity of those bearing a shorter linker. In particular, the apparent k cat of Rz3 increases by approximately 10-fold upon addition of saturating amounts of FMN. To determine the rate constants( K m4and k cat), the ribozyme regulated most effectively by FMN was further investigated. FMN mainly affected the k cat value, reflecting the rate limiting conformational change step of the overall cleavage reaction, depending on helix formation in stem II. Probably, FMN influences the orientation of structures necessary for the cleavage reaction through stem II formation. The result of chemical modification revealed that binding of FMN to the aptamer domain induced the helix formation in stem II required for catalytic activity. Therefore, a specific FMN-mediated allosteric interaction seems to promote a conformational alteration from an open to a closed structure in stem II. The concept of conformational modification in the allosteric effect is consistent with other allosteric enzymes, suggesting that such a conformational change is a fundamental feature of allosteric enzymes in biological systems.     

The catalytic core of RNase P.

A deletion mutant of the catalytic RNA component of Escherichia coli RNase P missing residues 87-241 retains the ability to interact with the protein component to form a functional catalyst. The deletion of this phylogenetically conserved region significantly increases the Km, indicating that the deleted structures may be important for binding to the precursor tRNA substrate but not for the cleavage reaction. Under some reaction conditions, this RNase P deletion mutant can become a relatively non-specific nuclease, indicating that this RNA's catalytic center may be more exposed. The catalytic core of the RNase P is formed by less than one third of the 377 residues of the RNase P RNA.     

Identification and characterization of a short 2′–3′ bond-forming ribozyme

A large number of natural and artificial ribozymes have been isolated since the demonstration of the catalytic potential of RNA, with the majority of these catalyzing phosphate hydrolysis or transesterification reactions. Here, we describe and characterize an extremely short ribozyme that catalyzes the positionally specific transesterification that produces a 2′–3′ phosphodiester bond between itself and a branch substrate provided in trans, cleaving itself internally in the process. Although this ribozyme was originally derived from constructs based on snRNAs, its minimal catalytic motif contains essentially no snRNA sequence and the reaction it catalyzes is not directly related to either step of pre-mRNA splicing. Our data have implications for the intrinsic reactivity of the large amount of RNA sequence space known to be transcribed in nature and for the validity and utility of the use of protein-free systems to study pre-mRNA splicing.     

RNA catalytic properties of the minimum (-)sTRSV sequence

We have identified an RNA catalytic domain within the sequence of the 359 base long negative-strand satellite RNA of tobacco ringspot virus. The catalytic domain contains two minimal sequences of satellite RNA, a 50-base catalytic RNA sequence, and a 14-base substrate RNA sequence. The catalytic complex of catalytic RNA/substrate RNA represents a structure not previously found in any RNA catalytic reaction described to date. The reaction is truly catalytic since the catalytic RNA has multiple substrate cleavage events and is not consumed during the course of the reaction. A linear relationship is seen between reaction rate and catalytic RNA concentration. The reaction has a Km of 0.03 microM, a kcat of 2.1/min, a temperature optimum of near 37 degrees C, and an energy of activation of 19 kcal/mol.     

Utilization of cofactors expands metabolism in a new RNA world

RNA has been hypothesized to have preceded proteins as the major catalysts of the biosphere, yet there are only a very limited number of chemical reactions that are known to be catalyzed by modern RNA. Cofactors are used by the majority of protein enzymes to supply additional functional groups to the active site. RNA should also be able to utilize some of these same cofactors to extend its own catalytic potential. We describe here how it could be possible to use selection — amplification from a population of random RNA to obtain a coenzyme A mediated RNA transacylase. Exploitation of some of the sulphur chemistry mediated by coenzyme A could have significantly expanded a prebiotic RNA directed metabolism.     

Design of allosteric hammerhead ribozymes activated by ligand-induced structure stabilization.

BACKGROUND: Ribozymes can function as allosteric enzymes that undergo a conformational change upon ligand binding to a site other than the active site. Although allosteric ribozymes are not known to exist in nature, nucleic acids appear to be well suited to display such advanced forms of kinetic control. Current research explores the mechanisms of allosteric ribozymes as well as the strategies and methods that can be used to create new controllable enzymes. RESULTS: In this study, we exploit the modular nature of certain functional RNAs to engineer allosteric ribozymes that are activated by flavin mononucleotide (FMN) or theophylline. By joining an FMN- or theophylline-binding domain to a hammerhead ribozyme by different stem II elements, we have identified a minimal connective bridge comprised of a G.U wobble pair that is responsive to ligand binding. Binding of FMN or theophylline to its allosteric site induces a conformational change in the RNA that stabilizes the wobble pair and ultimately favors the active form of the catalytic core. These ligand-sensitive ribozymes exhibit rate enhancements of more than 100-fold in the presence of FMN and of approximately 40-fold in the presence of theophylline. CONCLUSIONS: An adaptive strategy for modular rational design has proven to be an effective approach to the engineering of novel allosteric ribozymes. This strategy was used to create allosteric ribozymes that function by a mechanism involving ligand-induced structure stabilization. Conceivably, similar engineering strategies and allosteric mechanisms could be used to create a variety of novel allosteric ribozymes that function with other effector molecules.     

Initiation of translation with Pseudomonas aeruginosa phage PP7 RNA: nucleotide sequence of the coat cistron ribosome binding site.

Initiation complex formation between PP7 RNA and ribosomes of Pseudomonas aeruginosa and Escherichia coli has been investigated. The PP7 RNA fragments protected by both species of ribosome have been isolated, and their sequences have been determined. Only one binding sites is available on the intact PP7 RNA strand, and this site is recognized by ribosomes of both species. The PP7 RNA binding site is approximately 38 nucleotides long. It contains two AUG sequences and a purine-rich segment near the 5'-end that is complementary to segments near the 3'-ends of the 16S ribosomal RNA's of both P. aeruginosa and E. coli. In order to establish which of the AUG codons acts as the initiator, the H2N-terminal amino acid sequence of PP7 coat protein was determined. This sequence is compatible with the codon sequence following the second AUG codon. The extent of the reaction of PP7 RNA with E. coli ribosomes is greater than with P. aeruginosa ribosomes, but our results do not indicate a qualitative difference in the initial interaction between intact PP7 RNA and the ribosomes of either species.     

Computational design and experimental validation of oligonucleotide-sensing allosteric ribozymes

Allosteric RNAs operate as molecular switches that alter folding and function in response to ligand binding. A common type of natural allosteric RNAs is the riboswitch; designer RNAs with similar properties can be created by RNA engineering. We describe a computational approach for designing allosteric ribozymes triggered by binding oligonucleotides. Four universal types of RNA switches possessing AND, OR, YES and NOT Boolean logic functions were created in modular form, which allows ligand specificity to be changed without altering the catalytic core of the ribozyme. All computationally designed allosteric ribozymes were synthesized and experimentally tested in vitro. Engineered ribozymes exhibit >1,000-fold activation, demonstrate precise ligand specificity and function in molecular circuits in which the self-cleavage product of one RNA triggers the action of a second. This engineering approach provides a rapid and inexpensive way to create allosteric RNAs for constructing complex molecular circuits, nucleic acid detection systems and gene control elements.     

Mapping l1 ligase ribozyme conformational switch

L1 ligase (L1L) molecular switch is an in vitro optimized synthetic allosteric ribozyme that catalyzes the regioselective formation of a 5′-to-3′ phosphodiester bond, a reaction for which there is no known naturally occurring RNA catalyst. L1L serves as a proof of principle that RNA can catalyze a critical reaction for prebiotic RNA self-replication according to the RNA world hypothesis. L1L crystal structure captures two distinct conformations that differ by a reorientation of one of the stems by around 80 Å and are presumed to correspond to the active and inactive state, respectively. It is of great interest to understand the nature of these two states in solution and the pathway for their interconversion. In this study, we use explicit solvent molecular simulation together with a novel enhanced sampling method that utilizes concepts from network theory to map out the conformational transition between active and inactive states of L1L. We find that the overall switching mechanism can be described as a three‐state/two‐step process. The first step involves a large-amplitude swing that reorients stem C. The second step involves the allosteric activation of the catalytic site through distant contacts with stem C. Using a conformational space network representation of the L1L switch transition, it is shown that the connection between the three states follows different topographical patterns: the stem C swing step passes through a narrow region of the conformational space network, whereas the allosteric activation step covers a much wider region and a more diverse set of pathways through the network.