Probing the legitimate initiation of RNA synthesis by Qß replicase with oligonucleotide primers.

Oligoribonucleotides complementary to the template 3' terminus were tested for their ability to initiate RNA synthesis on legitimate templates capable of exponential amplification by Qß replicase. Oligonucleotides shorter than the distance to the nearest predicted template hairpin proved able to serve as primers, with the optimal length varying for different templates, suggesting that during initiation the template retains its native fold incorporating the 3' terminus. The priming activity of an oligonucleotide is greatly enhanced by its 5'-triphosphate group, the effect being strongly dependent on Mg2+ ions. This indicates that, unlike other studied RNA polymerases, Qß replicase binds the 5'-triphosphate of the initiating nucleotide GTP, and this binding is needed for the replication of legitimate templates.

Slippage at the initiation of RNA synthesis by Qß replicase results in a periodic polyG pattern.

The repetitive copying of template nucleotides due to transcriptional slippage has not been reported for RNA-directed RNA polymerases of positive-strand RNA phages. We unexpectedly observed that, with GTP as the only substrate, Qß replicase, the RNA-directed RNA polymerase of bacteriophage Qß, synthesizes by transcriptional slippage polyG strands, which on denaturing electrophoresis produce a ladder with at least three clusters of bolder bands. The ≈ 15-nt-long G15 , the major product of the shortest cluster, is tightly bound by the enzyme but can be released by the ribosomal protein S1, which, as a Qß replicase subunit, normally promotes the release of a completed transcript. 7-deaza-GTP suppresses the polyG synthesis and abolishes the periodic pattern, suggesting that the N7 atom is needed for the initiation of RNA synthesis and the formation of the structure recognized by protein S1. The results provide new insights into the mechanism of RNA synthesis by the RNA-directed RNA polymerase of a single-stranded RNA phage.

Structural transition of replicable RNAs during in vitro evolution with Qß replicase.

Single-stranded RNAs (ssRNAs) are utilized as genomes in some viruses and also in experimental models of ancient life-forms, owing to their simplicity. One of the largest problems for ssRNA replication is the formation of double-stranded RNA (dsRNA), a dead-end product for ssRNA replication. A possible strategy to avoid dsRNA formation is to create strong intramolecular secondary structures of ssRNA. To design ssRNAs that efficiently replicate by Qß replicase with minimum dsRNA formation, we previously proposed the "fewer unpaired GC rule." According to this rule, ssRNAs that have fewer unpaired G and C bases in the secondary structure should efficiently replicate with less dsRNA formation. However, the validity of this rule still needs to be examined, especially for longer ssRNAs. Here, we analyze nine long ssRNAs that successively appeared during an in vitro evolution of replicable ssRNA by Qß replicase and examine whether this rule can explain the structural transitions of the RNAs. We found that these ssRNAs improved their template abilities step-by-step with decreasing dsRNA formation as mutations accumulated. We then examine the secondary structures of all the RNAs by a chemical modification method. The analysis of the structures revealed that the probabilities of unpaired G and C bases tended to decrease gradually in the course of evolution. The decreases were caused by the local structural changes around the mutation sites in most of the cases. These results support the validity of the "fewer unpaired GC rule" to efficiently design replicable ssRNAs by Qß replicase, useful for more complex ssRNA replication systems.

A Direct RNA-to-RNA Replication System for Enhanced Gene Expression in Bacteria.

A long-standing objective of metabolic engineering has been to exogenously increase the expression of target genes. In this research, we proposed the permanent RNA replication system using DNA as a template to store genetic information in bacteria. We selected Qß phage as the RNA replication prototype and made many improvements to achieve target gene expression enhancement directly by increasing mRNA abundance. First, we identified the endogenous gene Rnc, the knockout of which significantly improved the RNA replication efficiency. Second, we elucidated the essential elements for RNA replication and optimized the system to make it more easily applicable. Combined with optimization of the host cell and the system itself, we developed a stable RNA-to-RNA replication tool to directly increase the abundance of the target mRNA and subsequently the target protein. Furthermore, it was proven efficient in enhancing the expression of specific proteins and was demonstrated to be applicable in metabolic engineering. Our system has the potential to be combined with any of the existing methods for increasing gene expression.

A Fusion Method to Develop an Expanded Artificial Genomic RNA Replicable by Qß Replicase.

RNA-based genomes are used to synthesize artificial cells that harbor genome replication systems. Previously, continuous replication of an artificial genomic RNA that encoded an RNA replicase was demonstrated. The next important challenge is to expand such genomes by increasing the number of encoded genes. However, technical difficulties are encountered during such expansions because the introduction of new genes disrupts the secondary structure of RNA and makes RNA nonreplicable through replicase. Herein, a fusion method that enables the construction of a longer RNA from two replicable RNAs, while retaining replication capability, is proposed. Two replicable RNAs that encode different genes at various positions are fused, and a new parameter, the unreplicable index, which negatively correlates with the replication ability of the fused RNAs better than that of the previous parameter, is determined. The unreplicable index represents the expected value of the number of G or C nucleotides that are unpaired in both the template and complementary strands. It is also observed that some of the constructed fused RNAs replicate efficiently by using the internally translated replicase. The method proposed herein could contribute to the development of an expanded RNA genome that can be used for the purpose of artificial cell synthesis.

Adaptation and Diversification of an RNA Replication System under Initiation- or Termination-Impaired Translational Conditions.

Adaptation to various environments is a remarkable characteristic of life. Is this limited to extant complex living organisms, or is it also possible for a simpler self-replication system to adapt? In this study, we addressed this question by using a translation-coupled RNA replication system that comprised a reconstituted translation system and an RNA "genome" that encoded a replicase gene. We performed RNA replication reactions under four conditions, under which different components of translation were partly inhibited. We found that replication efficiency increased with the number of rounds of replication under all the tested conditions. The types of dominant mutations differed depending on the condition, thus indicating that this simple system adapted to different environments in different ways. This suggests that even a primitive self-replication system composed of a small number of genes on the early earth could have had the ability to adapt to various environments.

Effect of Liposome Size on Internal RNA Replication Coupled with Replicase Translation.

Cell membranes inhibit the diffusion of intracellular materials, and compartment size can strongly affect the intracellular biochemical reactions. To assess the effect of the size of microcompartments on intracellular reactions, we constructed a primitive cell model consisting of giant liposomes and a translation-coupled RNA replication (TcRR) system. The RNA was replicated by Qß replicase, which was translated from the RNA in giant liposomes encapsulating the cell-free translation system. A reporter RNA encoding the antisense strand of ß-glucuronidase was introduced into the system to yield a TcRR read-out (green fluorescence). We demonstrate that TcRR was hardly detectable in larger liposomes (230 fL) but was more effective in smaller (7.7 fL) liposomes. Our experimental and theoretical results show that smaller microcompartments considerably enhance TcRR because the synthesized molecules, such as RNA and replicases, are more concentrated in smaller liposomes.

Structural basis for RNA-genome recognition during bacteriophage Qß replication.

Upon infection of Escherichia coli by bacteriophage Qß, the virus-encoded ß-subunit recruits host translation elongation factors EF-Tu and EF-Ts and ribosomal protein S1 to form the Qß replicase holoenzyme complex, which is responsible for amplifying the Qß (+)-RNA genome. Here, we use X-ray crystallography, NMR spectroscopy, as well as sequence conservation, surface electrostatic potential and mutational analyses to decipher the roles of the ß-subunit and the first two oligonucleotide-oligosaccharide-binding domains of S1 (OB1-2) in the recognition of Qß (+)-RNA by the Qß replicase complex. We show how three basic residues of the ß subunit form a patch located adjacent to the OB2 domain, and use NMR spectroscopy to demonstrate for the first time that OB2 is able to interact with RNA. Neutralization of the basic residues by mutagenesis results in a loss of both the phage infectivity in vivo and the ability of Qß replicase to amplify the genomic RNA in vitro. In contrast, replication of smaller replicable RNAs is not affected. Taken together, our data suggest that the ß-subunit and protein S1 cooperatively bind the (+)-stranded Qß genome during replication initiation and provide a foundation for understanding template discrimination during replication initiation.

Replication of partial double-stranded RNAs by Qß replicase.

Qß replicase, an RNA-dependent RNA polymerase of bacteriophage Qß, uses single-stranded RNA as a template to synthesize the complementary strand. A single-stranded RNA template may contain rigid secondary structures, such as long stems, intermolecular double-stranded RNA regions. Presently, the effect of the size of such double-stranded regions on the replication of RNA by Qß replicase is unknown. In this study, we prepared RNA templates hybridized with complementary RNA or DNA strands of various sizes and analyzed their replication by Qß replicase. We found that Qß replicase synthesizes the complementary strand as long as the template RNA is hybridized with no more than 200 nt fragments, although the replication amounts were decreased. This is important information to evaluate processivity of Qß replicase.

Structures and functions of Qß replicase: translation factors beyond protein synthesis.

Qß replicase is a unique RNA polymerase complex, comprising Qß virus-encoded RNA-dependent RNA polymerase (the catalytic ß-subunit) and three host-derived factors: translational elongation factor (EF) -Tu, EF-Ts and ribosomal protein S1. For almost fifty years, since the isolation of Qß replicase, there have been several unsolved, important questions about the mechanism of RNA polymerization by Qß replicase. Especially, the detailed functions of the host factors, EF-Tu, EF-Ts, and S1, in Qß replicase, which are all essential in the Escherichia coli (E. coli) host for protein synthesis, had remained enigmatic, due to the absence of structural information about Qß replicase. In the last five years, the crystal structures of the core Qß replicase, consisting of the ß-subunit, EF-Tu and Ts, and those of the core Qß replicase representing RNA polymerization, have been reported. Recently, the structure of Qß replicase comprising the ß-subunit, EF-Tu, EF-Ts and the N-terminal half of S1, which is capable of initiating Qß RNA replication, has also been reported. In this review, based on the structures of Qß replicase, we describe our current understanding of the alternative functions of the host translational elongation factors and ribosomal protein S1 in Qß replicase as replication factors, beyond their established functions in protein synthesis.

Changes in protein domains outside the catalytic site of the bacteriophage Qß replicase reduce the mutagenic effect of 5-azacytidine.

UNLABELLED: The high genetic heterogeneity and great adaptability of RNA viruses are ultimately caused by the low replication fidelity of their polymerases. However, single amino acid substitutions that modify replication fidelity can evolve in response to mutagenic treatments with nucleoside analogues. Here, we investigated how two independent mutants of the bacteriophage Qß replicase (Thr210Ala and Tyr410His) reduce sensitivity to the nucleoside analogue 5-azacytidine (AZC). Despite being located outside the catalytic site, both mutants reduced the mutation frequency in the presence of the drug. However, they did not modify the type of AZC-induced substitutions, which was mediated mainly by ambiguous base pairing of the analogue with purines. Furthermore, the Thr210Ala and Tyr410His substitutions had little or no effect on replication fidelity in untreated viruses. Also, both substitutions were costly in the absence of AZC or when the action of the drug was suppressed by adding an excess of natural pyrimidines (uridine or cytosine). Overall, the phenotypic properties of these two mutants were highly convergent, despite the mutations being located in different domains of the Qß replicase. This suggests that treatment with a given nucleoside analogue tends to select for a unique functional response in the viral replicase. IMPORTANCE: In the last years, artificial increase of the replication error rate has been proposed as an antiviral therapy. In this study, we investigated the mechanisms by which two substitutions in the Qß replicase confer partial resistance to the mutagenic nucleoside analogue AZC. As opposed to previous work with animal viruses, where different mutations selected sequentially conferred nucleoside analogue resistance through different mechanisms, our results suggest that there are few or no alternative AZC resistance phenotypes in Qß. Also, despite resistance mutations being highly costly in the absence of the drug, there was no sequential fixation of secondary mutations. Bacteriophage Qß is the virus with the highest reported mutation rate, which should make it particularly sensitive to nucleoside analogue treatments, probably favoring resistance mutations even if they incur high costs. The results are also relevant for understanding the possible pathways by which fidelity of the replication machinery can be modified.

Effects of ribosomes on the kinetics of Qß replication.

Bacteriophage Qß utilizes some host cell translation factors during replication. Previously, we constructed a kinetic model that explains replication of long RNA molecules by Qß replicase. Here, we expanded the previous kinetic model to include the effects of ribosome concentration on RNA replication. The expanded model quantitatively explained single- and double-strand formation kinetics during replication with various ribosome concentrations for two artificial long RNAs. This expanded model and the knowledge obtained in this study provide useful frameworks to understand the precise replication mechanism of Qß replicase with ribosomes and to design amplifiable RNA genomes in translation-coupling systems.

Characterization of norovirus RNA replicase for in vitro amplification of RNA.

BACKGROUND: The isothermal amplification of RNA in vitro has been used for the study of in vitro evolution of RNA. Although Qß replicase has been traditionally used as an enzyme for this purpose, we planned to use norovirus replicase (NV3D(pol)) due to its structural simplicity in the scope of in vitro autonomous evolution of the protein. Characteristics of the enzyme NV3D(pol) in vitro were re-evaluated in this context. RESULTS: NV3D(pol), synthesized by using a cell-free translation system, represented the activities which were reported in the previous several studies and the reports were not fully consistent each other. The efficiency of the initiation of replication was dependent on the 3'-terminal structure of single-stranded RNA template, and especially, NV3D(pol) preferred a self-priming small stem-loop. In the non-self-priming and primer-independent replication reaction, the presence of -CCC residues at the 3'-terminus increased the initiation efficiency and we demonstrated the one-pot isothermal RNA (even dsRNA) amplification by 16-fold. NV3D(pol) also showed a weak activity of elongation-reaction from a long primer. Based on these results, we present a scheme of the primer-independent isothermal amplification of RNA with NV3D(pol) in vitro. CONCLUSIONS: NV3D(pol) can be used as an RNA replicase in in vitro RNA + protein evolution with the RNA of special terminal sequences.

[Paradoxes of replication of RNA of a bacterial virus].

The extraordinary ability of the bacteriophage Qbeta replicase to amplify RNA outside the cell attracted attention of molecular biologists in the late 60's-early 70's. However, at that time, a number of puzzling properties of the enzyme did not received a rational explanation. Only recently, Qbeta-replicase began to uncover its secrets, promising to give a key not only to understanding the mechanism of replication of the genome of the bacterial virus, but also to the solution of more general fundamental and applied problems.

Identification of two forms of Q{beta} replicase with different thermal stabilities but identical RNA replication activity.

The enzyme Qß replicase is an RNA-dependent RNA polymerase, which plays a central role in infection by the simple single-stranded RNA virus bacteriophage Qß. This enzyme has been used in a number of applications because of its unique activity in amplifying RNA from an RNA template. Determination of the thermal stability of Qß replicase is important to gain an understanding of its function and potential applications, but data reported to date have been contradictory. Here, we provide evidence that these previous inconsistencies were due to the heterogeneous forms of the replicase with different stabilities. We purified two forms of replicase expressed in Escherichia coli, which differed in their thermal stability but showed identical RNA replication activity. Furthermore, we found that the replicase undergoes conversion between these forms due to oxidation, and the Cys-533 residue in the catalytic ß subunit and Cys-82 residue in the EF-Tu subunit of the replicase are essential prerequisites for this conversion to occur. These results strongly suggest that the thermal stable replicase contains the intersubunit disulfide bond between these cysteines. The established strategies for isolating and purifying a thermally stable replicase should increase the usefulness of Qß replicase in various applications, and the data regarding thermal stability obtained in this study may yield insight into the precise mechanism of infection by bacteriophage Qß.

Structure of the Qbeta replicase, an RNA-dependent RNA polymerase consisting of viral and host proteins.

The RNA-dependent RNA polymerase core complex formed upon infection of Escherichia coli by the bacteriophage Qbeta is composed of the viral catalytic beta-subunit as well as the host translation elongation factors EF-Tu and EF-Ts, which are required for initiation of RNA replication. We have determined the crystal structure of the complex between the beta-subunit and the two host proteins to 2.5-A resolution. Whereas the basic catalytic machinery in the viral subunit appears similar to other RNA-dependent RNA polymerases, a unique C-terminal region of the beta-subunit engages in extensive interactions with EF-Tu and may contribute to the separation of the transient duplex formed between the template and the nascent product to allow exponential amplification of the phage genome. The evolution of resistance by the host appears to be impaired because of the interactions of the beta-subunit with parts of EF-Tu essential in recognition of aminoacyl-tRNA.

Importance of translation-replication balance for efficient replication by the self-encoded replicase.

In all living systems, the genetic information is replicated by the self-encoded replicase (Rep); this can be said to be a self-encoding system. Recently, we constructed a self-encoding system in liposomes as an artificial cell model, consisting of a reconstituted translation system and an RNA encoding the catalytic subunit of Qbeta Rep and the RNA was replicated by the self-encoded Rep produced by the translation reaction. In this system, both the ribosome (Rib) and Rep bind to the same RNA for translation and replication, respectively. Thus, there could be a dilemma: effective RNA replication requires high levels of Rep translation, but excessive translation in turn inhibits replication. Herein, we actually observed the competition between the Rib and Rep, and evaluated the effect for RNA replication by constructing a kinetic model that quantitatively explained the behavior of the self-encoding system. Both the experimental and theoretical results consistently indicated that the balance between translation and replication is critical for an efficient self-encoded system, and we determined the optimum balance.

Functional circularity of legitimate Qbeta replicase templates.

Qbeta replicase (RNA-directed RNA polymerase of bacteriophage Qbeta) exponentially amplifies certain RNAs in vitro. Previous studies have shown that Qbeta replicase can initiate and elongate on a variety of RNAs; however, only a minute fraction of them are recognized as 'legitimate' templates. Guanosine 5'-triphosphate (GTP)-dependent initiation on a legitimate template generates a stable replicative complex capable of elongation in the presence of aurintricarboxylic acid, a powerful inhibitor of RNA-protein interactions. On the contrary, initiation on an illegitimate template is GTP independent and does not result in the aurintricarboxylic-acid-resistant replicative complex. This article demonstrates that the 3' and 5' termini of a legitimate template cooperate during and after the initiation step. Breach of the cooperation by dividing the template into fragments or by introducing point mutations at the 5' terminus reduces the rate and the yield of initiation, increases the GTP requirement, decreases the overall rate of template copying, and destabilizes the postinitiation replicative complex. These results revive the old idea of a functional circularity of legitimate Qbeta replicase templates and complement the increasing body of evidence that functional circularity may be a common property of RNA templates directing the synthesis of either RNA or protein molecules.