Template-directed RNA Synthesis Research Articles

Computer simulations of Template-Directed RNA Synthesis driven by temperature cycling in diverse sequence mixtures.

We present simulations of non-enzymatic template-directed RNA synthesis that incorporate primer extension, ligation, melting, and reannealing. Strand growth occurs over multiple heating/cooling cycles, producing strands of several hundred nucleotides in length, starting with random oligomers of 4 to 10 nucleotides. A strand typically grows by only 1 or 2 nucleotides in each cycle. Therefore, a strand is copied from many different templates, not from one specific complementary strand. A diverse sequence mixture is produced, and there is no exact copying of sequences, even if single base additions are fully accurate (no mutational errors). It has been proposed that RNA systems may contain a virtual circular genome, in which sequences partially overlap in a way that is mutually catalytic. We show that virtual circles do not emerge naturally in our simulations, and that a system initiated with a virtual circle can only maintain itself if there are no mutational errors and there is no input of new sequences formed by random polymerization. Furthermore, if a virtual sequence and its complement contain repeated short words, new sequences can be produced that were not on the original virtual circle. Therefore the virtual circle sequence cannot maintain itself. Functional sequences with secondary structures contain complementary words on opposite sides of stem regions. Both these words are repeated in the complementary sequence; hence, functional sequences cannot be encoded on a virtual circle. Additionally, we consider sequence replication in populations of protocells. We suppose that functional ribozymes benefit the cell which contains them. Nevertheless, scrambling of sequences occurs, and the functional sequence is not maintained, even when under positive selection.

Structure-Activity Relationships in Nonenzymatic Template-Directed RNA Synthesis.

The template-directed synthesis of RNA played an important role in the transition from prebiotic chemistry to the beginnings of RNA based life, but the mechanism of RNA copying chemistry is incompletely understood. We measured the kinetics of template copying with a set of primers with modified 3'-nucleotides and determined the crystal structures of these modified nucleotides in the context of a primer/template/substrate-analog complex. pH-rate profiles and solvent isotope effects show that deprotonation of the primer 3'-hydroxyl occurs prior to the rate limiting step, the attack of the alkoxide on the activated phosphate of the incoming nucleotide. The analogs with a 3 E ribose conformation show the fastest formation of 3'-5' phosphodiester bonds. Among those derivatives, the reaction rate is strongly correlated with the electronegativity of the 2'-substituent. We interpret our results in terms of differences in steric bulk and charge distribution in the ground vs. transition states.

The Enzymes of the Rifamycin Antibiotic Resistome.

Rifamycin antibiotics include the WHO essential medicines rifampin, rifabutin, and rifapentine. These are semisynthetic derivatives of the natural product rifamycins, originally isolated from the soil bacterium Amycolatopsis rifamycinica. These antibiotics are primarily used to treat mycobacterial infections, including tuberculosis. Rifamycins act by binding to the β-subunit of bacterial RNA polymerase, inhibiting transcription, which results in cell death. These antibiotics consist of a naphthalene core spanned by a polyketide ansa bridge. This structure presents a unique 3D configuration that engages RNA polymerase through a series of hydrogen bonds between hydroxyl groups linked to the naphthalene core and C21 and C23 of the ansa bridge. This binding occurs not in the enzyme active site where template-directed RNA synthesis occurs but instead in the RNA exit tunnel, thereby blocking productive formation of full-length RNA. In their clinical use to treat tuberculosis, resistance to rifamycin antibiotics arises principally from point mutations in RNA polymerase that decrease the antibiotic's affinity for the binding site in the RNA exit tunnel. In contrast, the rifamycin resistome of environmental mycobacteria and actinomycetes is much richer and diverse. In these organisms, rifamycin resistance includes many different enzymatic mechanisms that modify and alter the antibiotic directly, thereby inactivating it. These enzymes include ADP ribosyltransferases, glycosyltransferases, phosphotransferases, and monooxygenases.ADP ribosyltransferases catalyze group transfer of ADP ribose from the cofactor NAD+, which is more commonly deployed for metabolic redox reactions. ADP ribose is transferred to the hydroxyl linked to C23 of the antibiotic, thereby sterically blocking productive interaction with RNA polymerase. Like ADP ribosyltransferases, rifamycin glycosyl transferases also modify the hydroxyl of position C23 of rifamycins, transferring a glucose moiety from the donor molecule UDP-glucose. Unlike other antibiotic resistance kinases that transfer the γ-phosphate of ATP to inactivate antibiotics such as aminoglycosides or macrolides, rifamycin phosphotransferases are ATP-dependent dikinases. These enzymes transfer the β-phosphate of ATP to the C21 hydroxyl of the rifamycin ansa bridge. The result is modification of a critical RNA polymerase binding group that blocks productive complex formation. On the other hand, rifamycin monooxygenases are FAD-dependent enzymes that hydroxylate the naphthoquinone core. The result of this modification is untethering of the ansa chain from the naphthyl moiety, disrupting the essential 3D shape necessary for productive RNA polymerase binding and inhibition that leads to cell death.All of these enzymes have homologues in bacterial metabolism that either are their direct precursors or share common ancestors to the resistance enzyme. The diversity of these resistance mechanisms, often redundant in individual bacterial isolates, speaks to the importance of protecting RNA polymerase from these compounds and validates this enzyme as a critical antibiotic target.

Nanoscale Molecular Dynamics Simulation and Free Energy Decomposition of Hepatitis C Virus and its Interaction with GTP

Hepatitis C Virus (HCV) NS5B protein is a RNA-dependent RNA polymerase essential for replication of the viral RNA genome. Genome of HCV is an RNA molecule of approximately 9600 nucleotides, structured in a coding region that contains one large open reading frame (ORF), flanked by non-translated regions (NTR) at the 5’ and 3’ ends encoding a polyprotein precursor of about 3,000 amino acids. High concentrations of GTP (guanosine 5’-triphosphate) have been shown to stimulate de novo RNA synthesis by HCV NS5B. Molecular dynamics (MD) simulations have been performed on NS5B complex to investigate the stability and conformational changes with GTP. It is observed that the backbone atom of NS5B attains stability after 50ns. In vitro, NS5B has been shown to be capable of template-directed RNA synthesis on its own, requiring only divalent metals (magnesium or manganese) as cofactors. In addition, the MM-GBSA approach was introduced to analyze the interactions between inhibitors and NS5B.

A viscous solvent enables information transfer from gene-length nucleic acids in a model prebiotic replication cycle.

Many hypotheses concerning the nature of early life assume that genetic information was once transferred through the template-directed synthesis of RNA, before the emergence of coded enzymes. However, attempts to demonstrate enzyme-free, template-directed synthesis of nucleic acids have been limited by 'strand inhibition', whereby transferring information from a template strand in the presence of its complementary strand is inhibited by the stability of the template duplex. Here, we use solvent viscosity to circumvent strand inhibition, demonstrating information transfer from a gene-length template (>300 nt) within a longer (545 bp or 3 kb) duplex. These results suggest that viscous environments on the prebiotic Earth, generated periodically by water evaporation, could have facilitated nucleic acid replication-particularly of long, structured sequences such as ribozymes. Our approach works with DNA and RNA, suggesting that viscosity-mediated replication is possible for a range of genetic polymers, perhaps even for informational polymers that may have preceded RNA.

Nonenzymatic template-directed RNA synthesis inside model protocells.

Efforts to recreate a prebiotically plausible protocell, in which RNA replication occurs within a fatty acid vesicle, have been stalled by the destabilizing effect of Mg(2+) on fatty acid membranes. Here we report that the presence of citrate protects fatty acid membranes from the disruptive effects of high Mg(2+) ion concentrations while allowing RNA copying to proceed, while also protecting single-stranded RNA from Mg(2+)-catalyzed degradation. This combination of properties has allowed us to demonstrate the chemical copying of RNA templates inside fatty acid vesicles, which in turn allows for an increase in copying efficiency by bathing the vesicles in a continuously refreshed solution of activated nucleotides.

Fast and accurate nonenzymatic copying of an RNA-like synthetic genetic polymer.

Recent advances suggest that it may be possible to construct simple artificial cells from two subsystems: a self-replicating cell membrane and a self-replicating genetic polymer. Although multiple pathways for the growth and division of model protocell membranes have been characterized, no self-replicating genetic material is yet available. Nonenzymatic template-directed synthesis of RNA with activated ribonucleotide monomers has led to the copying of short RNA templates; however, these reactions are generally slow (taking days to weeks) and highly error prone. N3'-P5'-linked phosphoramidate DNA (3'-NP-DNA) is similar to RNA in its overall duplex structure, and is attractive as an alternative to RNA because the high reactivity of its corresponding monomers allows rapid and efficient copying of all four nucleobases on homopolymeric RNA and DNA templates. Here we show that both homopolymeric and mixed-sequence 3'-NP-DNA templates can be copied into complementary 3'-NP-DNA sequences. G:T and A:C wobble pairing leads to a high error rate, but the modified nucleoside 2-thiothymidine suppresses wobble pairing. We show that the 2-thiothymidine modification increases both polymerization rate and fidelity in the copying of a 3'-NP-DNA template into a complementary strand of 3'-NP-DNA. Our results suggest that 3'-NP-DNA has the potential to serve as the genetic material of artificial biological systems.

5 Efficient and high-fidelity copying of an RNA-like model prebiotic system

Nonenzymatic RNA replication would provide an important bridge to the RNA world. However, the demonstration of efficient and high-fidelity copying chemistry remains a great experimental challenge. It requires an efficient mechanism that can lead to both a high rate of polymerization and a high degree of fidelity in the copying chemistry. Previous experiments concerning nonenzymatic template-directed synthesis of RNA with activated monomers have led to the copying of short RNA templates, but these reactions are generally slow (taking days to weeks) and highly error-prone. Therefore, the ability to efficiently and accurately copy arbitrary template sequences remains frustratingly out of reach. N3′-P5′-linked phosphoramidate DNA is a highly reactive model for self-replicating genetic materials and has been used for studies of nonenzymatic RNA self-replication. It is also an excellent RNA mimic, due to its similar overall duplex structure, rigidity, and level of hydration (Tereshko, Gryaznov, & Egli, 1998). Our experiments show that the high reactivity imparted by the presence of an amino nucleophile allows rapid and efficient copying of all four nucleobases on both homopolymeric and mixed templates. On the other hand, G:T wobble pairing leads to a high error rate. We have, therefore, investigated the use of the modified nucleobase, 2-thio T (Ts) (Sintim & Kool, 2006), to suppress formation of the G:T wobble base-pair. Our results illustrate that the 2-thio modification can both increase polymerization rate and enhance fidelity in this self-replicating N3′-P5′-DNA system. These results suggest that this simple nucleobase modification may have played a role in primordial RNA (or proto-RNA) replication. In addition to suppressing the G:T mismatch, an additional benefit gained from its stronger base-pairing with A is that it also reduces A:C mismatch formation. Thus, simple modifications of nucleobases might provide a means of suppressing mismatches to yield better fidelity. Taken together, our results show that a high rate of polymerization and a high degree of fidelity are not mutually exclusive, but can be achieved simultaneously in nonenzymatic copying of N3′-P5′-linked phosphoramidate DNA. The structural similarity of NP-DNA to RNA suggests that these results could be translated to an RNA-only system.

The Mediator complex

The Mediator complex

Template‐Directed RNA Polymerization: The Taming of the Milieu

No-bias binding: The abiotic template-directed synthesis of RNA could have been a key process in the origins of life on Earth. Recreating this process in the laboratory has been challenging, yet a combination of strategies has given rise to a synthesis that is both efficient and unbiased against any of the four nucleotides.

Nucleotide synthetase ribozymes may have emerged first in the RNA world

Though the "RNA world" hypothesis has gained a central role in ideas concerning the origin of life, the scenario concerning its emergence remains uncertain. It has been speculated that the first scene may have been the emergence of a template-dependent RNA synthetase ribozyme, which catalyzed its own replication: thus, "RNA replicase." However, the speculation remains uncertain, primarily because of the large sequence length requirement of such a replicase and the lack of a convincing mechanism to ensure its self-favoring features. Instead, we propose a nucleotide synthetase ribozyme as an alternative candidate, especially considering recent experimental evidence suggesting the possibility of effective nonenzymatic template-directed synthesis of RNA. A computer simulation was conducted to support our proposal. The conditions for the emergence of the nucleotide synthetase ribozyme are discussed, based on dynamic analysis on a computer. We suggest the template-dependent RNA synthetase ribozyme emerged later, perhaps after the emergence of protocells.

Nonenzymatic Ligation of Short‐Chained 2′–5′‐ or 3′–5′‐Linked Oligoribonucleotides on 2′–5′‐ or 3′–5′‐Linked Complementary Templates

5'-pACUG tetraribonucleotides containing 2'-5' or 3'-5' linkages self-condensed on 2'-5'- or 3'-5'-linked complementary decaribonucleotide (5'-CAGUCAGUCA) templates. CD and UV melting studies showed that helix formation took place in all four possible combinations of linkage isomers of the substrate tetramer and the template decamer under the ligation conditions. The hybridization ability followed the order: [3'-5' tetramer with 3'-5' decamer] > [2'-5' tetramer with 3'-5' decamer] > [2'-5' tetramer with 2'-5' decamer] > or = [3'-5' tetramer with 2'-5' decamer]. Each tetramer condensed on the complementary decaribonucletide template to form the corresponding octamer, but the ligation efficiency varied considerably, depending on the types of linkage in the tetramer substrate and the template decamer. The yields of the octamers obtained by the template-directed ligation followed the order: [2'-5' substrate: 2'-5' template] > [3'-5':3'-5'] > [2'-5':3'-5'] > [3'-5':2'-5']. The results demonstrate that a homo-linkage system is preferable for the template-directed synthesis of RNA. The resulting linkage of the octamer formed from the 2'-5'-linked substrate and the 2'-5'-linked template is mainly 2'-5'.

Nonenzymatic template-directed RNA synthesis.

The aim of this chapter is to provide reliable protocols for the study of nonenzymatic oligomerization reactions of ribonucleic acid and its analogs. Traditional radioactive labels are replaced by fluorescent dyes. Product analysis is either based on reversed-phase high-pressure (performance) liquid chromatography or on gel electrophoresis with on-line detection applying a deoxyribonucleic acid sequencer. Three examples of primer extensions are given, one of which demonstrates how the reaction of several primers may be monitored simultaneously.

Expression of Functional Influenza Virus RNA Polymerase in the Methylotrophic Yeast Pichia pastoris

Influenza virus RNA polymerase with the subunit composition PB1-PB2-PA is a multifunctional enzyme with the activities of both synthesis and cleavage of RNA and is involved in both transcription and replication of the viral genome. In order to produce large amounts of the functional viral RNA polymerase sufficient for analysis of its structure-function relationships, the cDNAs for RNA segments 1, 2, and 3 of influenza virus A/PR/8, each under independent control of the alcohol oxidase gene promoter, were integrated into the chromosome of the methylotrophic yeast Pichia pastoris. Simultaneous expression of all three P proteins in the yeast P. pastoris was achieved by the addition of methanol. To purify the P protein complexes, a sequence coding for a histidine tag was added to the PB2 protein gene at its N terminus. Starting from the induced P. pastoris cell lysate, we partially purified a 3P complex by Ni(2+)-agarose affinity column chromatography. The 3P complex showed influenza virus model RNA-directed and ApG-primed RNA synthesis in vitro but was virtually inactive without addition of template or primer. The kinetic properties of model template-directed RNA synthesis and the requirements for template sequence were analyzed using the 3P complex. Furthermore, the 3P complex showed capped RNA-primed RNA synthesis. Thus, we conclude that functional influenza virus RNA polymerase with the catalytic properties of a transcriptase is formed in the methylotrophic yeast P. pastoris.

Nonenzymatic Template-directed Synthesis of RNA from Monomers

Reviewed are the latest achievements in studying the information transfer mechanisms and the evolutionary significance of prebiotic RNA synthesis, the double helix structures most preferred in this respect, and the possible reasons for the prevalence of particular enantiomeric forms of nucleotides in template-directed synthesis.

Influenza virus PB1 protein is the minimal and essential subunit of RNA polymerase.

RNA polymerase of influenza virus with the subunit structure PB1-PB2-PA is involved in both transcription and replication of the genome RNA. The RNA polymerase with transcription activity was reconstituted from three P proteins, which were separately isolated from insect cells infected with recombinant baculoviruses, each carrying cDNA for one P protein. Nuclear extracts of the insect cells infected with each of the recombinant baculoviruses or various combinations of these viruses were examined for transcription and replication activities. The nuclear extract of cells expressing all three P proteins catalyzed model template-directed RNA synthesis in the absence of primers (an indication of RNA replication), supporting the notion that the complete set of three P proteins is required for RNA replication. All the nuclear extracts containing the PB1 subunit, including the extract containing PB1 alone, were able to catalyze model template-directed dinucleotide-primed RNA synthesis (an indication of transcription). These observations not only confirm that the PB1 protein is a catalytic subunit of influenza virus RNA polymerase, but also indicate that PB1 alone is able to catalyze RNA synthesis in the absence of PB2 and PA subunits.

RNA template-directed RNA synthesis by T7 RNA polymerase.

In an attempt to synthesize an oligoribonucleotide by run-off transcription by bacteriophage T7 RNA polymerase, a major transcript was produced that was much longer than expected. Analysis of the reaction indicated that the product resulted from initial DNA-directed run-off transcription followed by RNA template-directed RNA synthesis. This reaction occurred because the RNA made from the DNA template displayed self-complementarity at its 3' end and therefore could form an intra- or intermolecular primed template. In reactions containing only an RNA template, the rate of incorporation of NTPs was quite comparable to DNA-dependent transcription. RNA template-directed RNA synthesis has been found to occur with a great number of oligoribonucleotides, even with primed templates that are only marginally stable. In one instance, we observed a multistep extension reaction converting the oligonucleotide into a final product longer than twice its original length. Presumably, such a process could have generated some of the RNAs found to be efficiently replicated by T7 RNA polymerase.

Chiral selection in template-directed RNA synthesis and the case for a prochiral ancestor of RNA

Poly(C)-directed oligomerization of activated guanosine mononucleotides proceeds readily if the monomers are of the same optical handedness as the template, but is far less efficient if the monomers are of the opposite handedness. In template-directed reactions with a racemic mixture, substantial enantiomeric cross-inhibition occurs. This presents an important problem for theories of the origin of life that are based on a system of self-replicating polynucleotides.

Evidence for de novo production of self-replicating and environmentally adapted RNA structures by bacteriophage Qbeta replicase.

Highly purified coliphage Qbeta replicase when incubated without added template synthesizes self-replicating RNA species in an autocatalytic reaction. In this paper we offer strong evidence that this RNA production is directed by templates generated de novo during the lag phase. Contamination of the enzyme by traces of RNA templates was ruled out by the following experimental results: (1) Additional purification steps do not eliminate this RNA production. (2) The lag phase is lengthened to several hours by lowering substrate or enzyme concentration. At a nucleoside triphosphate concentration of 0.15 mM no RNA is produced although the template-directed RNA synthesis works normally. (3) Different enzyme concentrations lead to RNA species of completely different primary structure. (4) Addition of oligonucleotides or preincubation with only three nucleoside triphosphates affects the final RNA sequence. (5) Manipulation of conditions during the lag phase results in the production of RNA structures that are adapted to the particular incubation conditions applied (e.g., RNA resistant to nuclease attack or resistant to inhibitors or even RNAs "addicted to the drug," in the sense that they only replicate in the presence of a drug like acridine orange). RNA species obtained in different experiments under optimal incubation conditions show very similar fingerprint patterns, suggesting the operation of an instruction mechanism. A possible mechanism is discussed.