Is your ribozyme design really correct?: A proposal of simple single turnover competition assay to evaluate ribozymes

Today, many nucleic acid enzymes are used in gene therapy and gene regulations. However, no simple assay methods to evaluate enzymatic activities, with which we judge the enzyme design, have been reported. Here, we propose a new simple competition assay for nucleic acid enzymes of different types to evaluate the cleaving efficiency of a target RNA molecule, of which the recognition sites are different but overlapped. Two nucleic acid enzymes were added to one tube to make a competition of these two enzymes for one substrate. The assay was used on two ribozymes, hammerhead ribozyme and hairpin ribozyme, and a DNA-enzyme. We found that this assay method is capable of application to those enzymes, as a powerful tool for the selection and designing of RNA-cleaving enzymes.     

Bioenergetics and Life's Origins

Bioenergetics is central to our understanding of living systems, yet has attracted relatively little attention in origins of life research. This article focuses on energy resources available to drive primitive metabolism and the synthesis of polymers that could be incorporated into molecular systems having properties associated with the living state. The compartmented systems are referred to as protocells, each different from all the rest and representing a kind of natural experiment. The origin of life was marked when a rare few protocells happened to have the ability to capture energy from the environment to initiate catalyzed heterotrophic growth directed by heritable genetic information in the polymers. This article examines potential sources of energy available to protocells, and mechanisms by which the energy could be used to drive polymer synthesis.Previous research on life''s origins has for the most part focused on the chemistry and energy sources required to produce the small molecules of life—amino acids, nucleobases, and amphiphiles—and to a lesser extent on condensation reactions by which the monomers can be linked into biologically relevant polymers. In modern living cells, polymers are synthesized from activated monomers such as the nucleoside triphosphates used by DNA and RNA polymerases, and the tRNA-amino acyl conjugates that supply ribosomes with activated amino acids. Activated monomers are essential because polymerization reactions occur in an aqueous medium and are therefore energetically uphill in the absence of activation.A central problem therefore concerns mechanisms by which prebiotic monomers could have been activated to assemble into polymers. Most biopolymers of life are synthesized when the equivalent of a water molecule is removed to form the ester bonds of nucleic acids, glycoside bonds of polysaccharides, and peptide bonds in proteins. In life today, the removal of water is performed upstream of the actual bond formation. This process involves the energetically downhill transfer of electrons, which is coupled to either substrate-level oxidation or generation of a proton gradient that in turn is the energy source for the synthesis of anhydride pyrophosphate bonds in ATP. The energy stored in the pyrophosphate bond is then distributed throughout the cell to drive most other energy‐dependent reactions. This is a complex and highly evolved process, so here we consider simpler ways in which energy could have been captured from the environment and made available for primitive versions of metabolism and polymer synthesis. Because the atmosphere of the primitive Earth did not contain appreciable oxygen, this review of primitive bioenergetics is limited to anaerobic sources of energy.     

Pyrophosphorolysis of CCA addition: implication for fidelity

In nucleic acid polymerization reaction, pyrophosphorolysis is the reversal of nucleotide addition, in which the terminal nucleotide is excised in the presence of inorganic pyrophosphate (PPi). The CCA enzymes are unusual RNA polymerases, which catalyze CCA addition to positions 74-76 at the tRNA 3′ end without using a nucleic acid template. To better understand the reaction mechanism of CCA addition, we tested pyrophosphorolysis of CCA enzymes, which are divided into two structurally distinct classes. Here, we show that only class II CCA enzymes catalyze pyrophosphorolysis and that the reaction can initiate from all three CCA positions and proceed processively until the removal of nucleotide C74. Pyrophosphorolysis of class II enzymes establishes a fundamental difference from class I enzymes, and it is achieved only with the tRNA structure and with specific divalent metal ions. Importantly, pyrophosphorolysis enables class II enzymes to efficiently remove an incorrect A75 nucleotide from the 3′ end, at a rate much faster than the rate of A75 incorporation, suggesting the ability to perform a previously unexpected quality control mechanism for CCA synthesis. Measurement of kinetic parameters of the class II Escherichia coli CCA enzyme reveals that the enzyme catalyzes pyrophosphorolysis slowly relative to the forward nucleotide addition and that it exhibits weak binding affinity to PPi relative to NTP, suggesting a mechanism in which PPi is rapidly released after each nucleotide addition as a driving force to promote the forward synthesis of CCA.     

Nucleic acid unwinding by hepatitis C virus and bacteriophage t7 helicases is sensitive to base pair stability

Helicases are motor enzymes that convert the chemical energy of NTP hydrolysis into mechanical force for motion and nucleic acid strand separation. Within the cell, helicases process a range of nucleic acid sequences. It is not known whether this composite rate of moving and opening the strands of nucleic acids depends on the base sequence. Our presteady state kinetic studies of helicases from two classes, the ring-shaped T7 helicase and two forms of non-ring-shaped hepatitis C virus (HCV) helicase, show that both the unwinding rate and processivity depend on the sequence and decrease as the nucleic acid stability increases. The DNA unwinding activity of T7 helicase and the RNA unwinding activity of HCV helicases decrease steeply with increasing base pair stability. On the other hand, the DNA unwinding activity of HCV helicases is less sensitive to base pair stability. These results predict that helicases will fall into a spectrum of modest to high sensitivity to base pair stability depending on their biological role in the cell. Modeling of the dependence provided the degree of the active involvement of helicase in base pair destabilization during the unwinding process and distinguished between passive and active mechanisms of unwinding.     

The crystal structure of human geranylgeranyl pyrophosphate synthase reveals a novel hexameric arrangement and inhibitory product binding

Modification of GTPases with isoprenoid molecules derived from geranylgeranyl pyrophosphate or farnesyl pyrophosphate is an essential requisite for cellular signaling pathways. The synthesis of these isoprenoids proceeds in mammals through the mevalonate pathway, and the final steps in the synthesis are catalyzed by the related enzymes farnesyl pyrophosphate synthase and geranylgeranyl pyrophosphate synthase. Both enzymes play crucial roles in cell survival, and inhibition of farnesyl pyrophosphate synthase by nitrogen-containing bisphosphonates is an established concept in the treatment of bone disorders such as osteoporosis or certain forms of cancer in bone. Here we report the crystal structure of human geranylgeranyl pyrophosphate synthase, the first mammalian ortholog to have its x-ray structure determined. It reveals that three dimers join together to form a propeller-bladed hexameric molecule with a mass of approximately 200 kDa. Structure-based sequence alignments predict this quaternary structure to be restricted to mammalian and insect orthologs, whereas fungal, bacterial, archaeal, and plant forms exhibit the dimeric organization also observed in farnesyl pyrophosphate synthase. Geranylgeranyl pyrophosphate derived from heterologous bacterial expression is tightly bound in a cavity distinct from the chain elongation site described for farnesyl pyrophosphate synthase. The structure most likely represents an inhibitory complex, which is further corroborated by steady-state kinetics, suggesting a possible feedback mechanism for regulating enzyme activity. Structural comparisons between members of this enzyme class give deeper insights into conserved features important for catalysis.     

Transketolase: observations in alcohol-related brain damage research

Thiamin, or vitamin B1, is crucial for brain function. In its active form, thiamin pyrophosphate (TPP), it is a co-enzyme for several enzymes, including transketolase. Transketolase is an important enzyme in the non-oxidative branch of the pentose phosphate pathway (PPP), a pathway responsible for generating reducing equivalents, which is essential for energy transduction and for generating ribose for nucleic acid synthesis. Transketolase also links the PPP to glycolysis, allowing a cell to adapt to a variety of energy needs, depending on its environment. Abnormal transketolase expression and/or activity have been implicated in a number of diseases where thiamin availability is low, including Wernicke-Korsakoff's Syndrome and alcoholism. Yet, the precise mechanism by which this enzyme is involved in the pathophysiology of these disorders remains controversial.     

Regulation of Synthesis of Two Immunologically Distinct Nucleic Acid-Dependent Nucleoside Triphosphate Phosphohydrolases in Vaccinia Virus-Infected HeLa Cells

The two nucleic acid-dependent nucleoside triphosphate phosphohydrolases, previously purified from vaccinia virus cores, were shown to be immunologically distinct enzymes. Antiserum prepared against purified phosphohydrolase I and antiserum prepared against purified phosphohydrolase II only neutralized the activity of that enzyme used as antigen. Both enzymes were induced in HeLa cells after vaccinia infection. DNA-cellulose chromatography was used to purify the two phosphohydrolases from the cytoplasms of infected cells. The enzymes were identified by their different substrate specificities, nucleic acid dependence, and neutralization with specific antiserum. A third chromatographically separable nucleic acid-dependent phosphohydrolase similar to phosphohydrolase I in substrate specificity but not neutralizable by antiserum to either phosphohydrolase I or II, was also isolated from infected cells. No nucleic acid-dependent nucleoside triphosphate phosphohydrolase activity was detected by similar methods from uninfected HeLa cells. Formation of these virus-induced enzymes was prevented by actinomycin D and cycloheximide, indicating a requirement for de novo RNA and protein synthesis, respectively. The kinetics of induction and inhibition by cytosine arabinoside, an inhibitor of DNA synthesis, suggested that synthesis of the phosphohydrolases is a late viral function. Rifampin, an inhibitor of vaccinia virus growth which prevents virion assembly, had no inhibitory effect on the induction of the phosphohydrolases. This result was consistent with the finding that these enzymes exist in a soluble as well as in a particulate form in the cytoplasm of infected cells. Addition of another specific anti-poxviral drug, isatin-beta-thiosemicarbazone, to vaccinia-infected cells partially inhibited induction of the phosphohydrolases.     

Characterization of replication fork and phosphorylation stimulated Plasmodium falciparum helicase 45

Helicases are essential enzymes, which play important role in the metabolism of nucleic acids. In the present study we report further characterization of PfH45 (Plasmodium falciparum helicase 45), which is an essential enzyme for parasite survival. The results show that the helicase activity of PfH45 is significantly stimulated by replication fork like structure. The studies using truncated derivatives of PfH45 show that its nucleic acid dependent ATPase activity resides in the N-terminal one third of the protein and its RNA and DNA-binding activity predominantly resides in the C-terminal two third of the protein. The phosphorylation of PfH45 by protein kinase C at Ser and Thr residues stimulated its DNA and RNA helicase and ssDNA and RNA-dependent ATPase activities. DNA-interacting compounds actinomycin, DAPI, daunorubicin, ethidium bromide, netropsin and nogalamycin were able to inhibit the helicase and ssDNA-dependent ATPase activity with apparent IC50 values ranging from 0.5 to 5.0 microM respectively. These compounds distinctively inhibit the helicase activity probably by forming complex with DNA and obstructing enzyme movement.     

Crystal structures of undecaprenyl pyrophosphate synthase in complex with magnesium, isopentenyl pyrophosphate, and farnesyl thiopyrophosphate: roles of the metal ion and conserved residues in catalysis

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes the consecutive condensation reactions of a farnesyl pyrophosphate (FPP) with eight isopentenyl pyrophosphates (IPP), in which new cis-double bonds are formed, to generate undecaprenyl pyrophosphate that serves as a lipid carrier for peptidoglycan synthesis of bacterial cell wall. The structures of Escherichia coli UPPs were determined previously in an orthorhombic crystal form as an apoenzyme, in complex with Mg(2+)/sulfate/Triton, and with bound FPP. In a further search of its catalytic mechanism, the wild-type UPPs and the D26A mutant are crystallized in a new trigonal unit cell with Mg(2+)/IPP/farnesyl thiopyrophosphate (an FPP analogue) bound to the active site. In the wild-type enzyme, Mg(2+) is coordinated by the pyrophosphate of farnesyl thiopyrophosphate, the carboxylate of Asp(26), and three water molecules. In the mutant enzyme, it is bound to the pyrophosphate of IPP. The [Mg(2+)] dependence of the catalytic rate by UPPs shows that the activity is maximal at [Mg(2+)] = 1 mm but drops significantly when Mg(2+) ions are in excess (50 mm). Without Mg(2+), IPP binds to UPPs only at high concentration. Mutation of Asp(26) to other charged amino acids results in significant decrease of the UPPs activity. The role of Asp(26) is probably to assist the migration of Mg(2+) from IPP to FPP and thus initiate the condensation reaction by ionization of the pyrophosphate group from FPP. Other conserved residues, including His(43), Ser(71), Asn(74), and Arg(77), may serve as general acid/base and pyrophosphate carrier. Our results here improve the understanding of the UPPs enzyme reaction significantly.     

Stimulation of nucleic acid biosynthesis by phosphopentoses

Phosphopentose stimulation of nucleic acids biosynthesis for 3h after subcutaneous phosphopentose administration in doses of 18 and 27 mg per rat has been stated. Injections of phosphopentoses (ribose-5-phosphate, xylulose-6-phosphate, ribulose-5-phosphate in the ratio of 1.0:0.3:0.3) were followed by a two-fold increase in the rate of [2-14C] orothic acid incorporation into cytoplasmic RNA of the rat liver. It is supposed that rapidly exchanging types of RNA contribute most of all to the effect of the label incorporation increase and the stimulation mechanism is associated with a rise of phosphoribosyl pyrophosphate accessibility as a substrate and an allosteric regulator of key enzymes of the synthesis of purine and pyrimidine nucleotides.     

Bacteriophage T4 RNA ligase. Reaction intermediates and interaction of substrates.

Bacteriophage T4 RNA ligase catalyzes the ATP-dependent ligation of a 5'-phosphoryl-terminated nucleic acid donor to a 3'-hydroxyl-terminated nucleic acid acceptor. We have identified adenylylated DNA and RNA reaction intermediates in which the AMP moiety is attached by a pyrophosphate bond to the 5'-phosphoryl group of the donor. A large amount of DNA-adenylate accumulates during the reaction and the dependence of joining and adenylylation on chain length are similar. The adenylylated donor is joined by ligase to an acceptor in the absence of ATP, and AMP is released stoichiometrically in this reaction. The acceptor is not only a substrate in the reaction but also a cofactor for adenylylation of the donor; in the absence of a 3'-hydroxyl group the activated intermediate does not form. The activated DNA need not join to the acceptor that initially stimulated activation but can also join to another acceptor. This process of acceptor exchanges has proven useful for promoting the cyclization of small DNA substrates and the synthesis of DNA co-polymers.     

Transliteration of synthetic genetic enzymes

Functional nucleic acids lose activity when their sequence is prepared in the backbone architecture of a different genetic polymer. The only known exception to this rule is a subset of aptamers whose binding mechanism involves G-quadruplex formation. We refer to such examples as transliteration—a synthetic biology concept describing cases in which the phenotype of a nucleic acid molecule is retained when the genotype is written in a different genetic language. Here, we extend the concept of transliteration to include nucleic acid enzymes (XNAzymes) that mediate site-specific cleavage of an RNA substrate. We show that an in vitro selected 2′-fluoroarabino nucleic acid (FANA) enzyme retains catalytic activity when its sequence is prepared as α-l-threofuranosyl nucleic acid (TNA), and vice versa, a TNA enzyme that remains functional when its sequence is prepared as FANA. Structure probing with DMS supports the hypothesis that FANA and TNA enzymes having the same primary sequence can adopt similarly folded tertiary structures. These findings provide new insight into the sequence-structure-function paradigm governing biopolymer folding.     

A continuous kinetic assay for adenylation enzyme activity and inhibition

Adenylation/adenylate-forming enzymes catalyze the activation of a carboxylic acid at the expense of ATP to form an acyl-adenylate intermediate and pyrophosphate (PP_i). In a second half-reaction, adenylation enzymes catalyze the transfer of the acyl moiety of the acyl-adenylate onto an acceptor molecule, which can be either a protein or a small molecule. We describe the design, development, and validation of a coupled continuous spectrophotometric assay for adenylation enzymes that employs hydroxylamine as a surrogate acceptor molecule, leading to the formation of a hydroxamate. The released pyrophosphate from the first half-reaction is measured using the pyrophosphatase-purine nucleoside phosphorylase coupling system with the chromogenic substrate 7-methylthioguanosine (MesG). The coupled hydroxamate-MesG assay is especially useful for characterizing the activity and inhibition of adenylation enzymes that acylate a protein substrate and/or fail to undergo rapid ATP-PP_i exchange.     

SYNTHETIC ACTIVITIES DURING SPERMATOGENESIS IN THE LOCUST

Isolated testes of the locust Schistocerca gregaria were immersed in solutions of tritiated thymidine, cytidine, uridine, or arginine for short periods to study nucleic acid and protein synthesis during spermatogenesis. DNA synthesis in this tissue is completed prior to initiation of meiosis. Protein synthesis continues throughout the whole meiotic cycle as well as during spermatid development. Meiotic cells, except those in metaphase through early telophase, and early spermatids are also actively synthesizing RNA. The heteropycnotic X-chromosome does not produce RNA at any stage of spermatogenesis. The rates of protein and particularly RNA synthesis decrease as chromosome condensation progresses. Depression of RNA synthesis, however, is not always accompanied by cytologically detectable condensation of chromatin, since very little or no RNA is synthesized in spermatids in which chromatin condensation has barely begun.     

Purification and characterization of palmitoyl-CoA ligase from rat brain microsomes

We have previously shown the existence of two separate enzymes for the synthesis of palmitoyl-CoA and lignoceroyl-CoA in rat brain microsomal membranes (1). Palmitoyl-CoA ligase activity was solubilized from brain microsomal membranes with 0.3% Triton X-100 and purified 93-fold by a combination of protein purification techniques. The Km values for the substrates palmitic acid, CoASH and ATP were 11.7 microM, 5.88 microM and 3.77 mM respectively. During activation of palmitic acid ATP is hydrolyzed to AMP and pyrophosphate, as evidenced by the inhibition of this activation by 5 mM concentrations of AMP, pyrophosphate or AMP and pyrophosphate to 70%, 60% and 85% respectively. The divalent metal ion Mg2+ was required for activity; its replacement with Mn2+ resulted in a 35% decrease in activity. Palmitoyl-CoA ligase activity was inhibited by the addition of oleic or stearic acids whereas addition of lignoceric acid or behenic acid had no effect. This supports our previous observation that palmitoyl-CoA and lignoceroyl-CoA are synthesized by two different enzymes in rat brain microsomal membranes.