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.

In vitro characterisation of the MS2 RNA polymerase complex reveals host factors that modulate emesviral replicase activity.

The RNA phage MS2 is one of the most important model organisms in molecular biology and virology. Despite its comprehensive characterisation, the composition of the RNA replication machinery remained obscure. Here, we characterised host proteins required to reconstitute the functional replicase in vitro. By combining a purified replicase sub-complex with elements of an in vitro translation system, we confirmed that the three host factors, EF-Ts, EF-Tu, and ribosomal protein S1, are part of the active replicase holocomplex. Furthermore, we found that the translation initiation factors IF1 and IF3 modulate replicase activity. While IF3 directly competes with the replicase for template binding, IF1 appears to act as an RNA chaperone that facilitates polymerase readthrough. Finally, we demonstrate in vitro formation of RNAs containing minimal motifs required for amplification. Our work sheds light on the MS2 replication machinery and provides a new promising platform for cell-free evolution.

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.

[Unsolved Puzzles of Qß Replicase].

Qß phage replicase has been the first RNA-directed RNA polymerase purified to homogeneity and intensively studied in vitro. In the mid-sixties, papers on Qß and related replicases appeared in nearly every issue of the PNAS journal. By 1968, the mechanism of its action seemed to be almost completely understood. However, even now, a half of century later, a number of fundamental questions remains unanswered. How does the replicase manage to prevent the template and its complementary copy from annealing during the entire replication round? How does it recognize its templates? What is the function of the translation factors present in the replicase molecule? What is the mechanism the replicase uses to join (recombine) separate RNA molecules? Even the determination of the crystal structure of Qß replicase did not help much. Certainly, there remains a lot to discover in the replication of Qß phage, one of the smallest viruses known.

Thirty Years of Studies of Qß Replicase: What Have We Learned and What Is Yet to Be Learned?

Qß replicase (RNA-directed RNA polymerase of bacteriophage Qß) has an unsurpassed capacity to amplify polynucleotides in vitro. In 1986, the Group of Viral RNA Biochemistry was organized at the Institute of Protein Research in order to exploit this property for the synthesis of messenger RNAs to be used in cell-free translation systems. Although the task has not been implemented in full, this work has led to a number of unexpected important results including uncovering the nature of the "template-free" RNA synthesis by Qß replicase, discovering the ability of RNA molecules for spontaneous recombination, revealing the unusual mechanism Qß replicase uses to discriminate between its proper and improper templates, and discovering a new function of the largest ribosomal protein S1, that is also one of the replicase subunits. Finally, our work resulted in the invention of the molecular colonies technique that has become the basis for the next generation sequencing methods and provided a new insight into the origin of life. However, Qß replicase has not yet revealed all its secrets, and its studies promise further interesting findings.

Combinatorial selection for replicable RNA by Qß replicase while maintaining encoded gene function.

Construction of a complex artificial self-replication system is challenging in the field of in vitro synthetic biology. Recently, we developed a translation-coupled RNA replication system, wherein an artificial genomic RNA replicates with the Qß RNA replicase gene encoded on itself. The challenge is to introduce additional genes into the RNA to develop a complex system that mimics natural living systems. However, most RNA sequence encoding genes are not replicable by the Qß replicase owing to its requirement for strong secondary structures throughout the RNA sequence that are absent in most genes. In this study, we establish a new combinatorial selection method to find an RNA sequence with secondary structures and functional amino acid sequences of the encoded gene. We selected RNA sequences based on their in vitro replication and in vivo gene functions. First, we used the α-domain gene of ß-galactosidase as a model-encoding gene, with functional selection based on blue-white screening. Through the combinatorial selection, we developed more replicable RNAs while maintaining the function of the encoded α-domain. The selected sequence improved the affinity between the minus strand RNA and Qß replicase. Second, we established an in vivo selection method applicable to a broader range of genes by using an Escherichia coli strain with one of the essential genes complemented with a plasmid. We performed the combinatorial selection using an RNA encoding serS and obtained more replicable RNA encoding functional serS gene. These results suggest that combinatorial selection methods are useful for the development of RNA sequences replicable by Qß replicase while maintaining the encoded gene function.

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.

Molecular insights into replication initiation by Qß replicase using ribosomal protein S1.

Ribosomal protein S1, consisting of six contiguous OB-folds, is the largest ribosomal protein and is essential for translation initiation in Escherichia coli. S1 is also one of the three essential host-derived subunits of Qß replicase, together with EF-Tu and EF-Ts, for Qß RNA replication in E. coli. We analyzed the crystal structure of Qß replicase, consisting of the virus-encoded RNA-dependent RNA polymerase (ß-subunit), EF-Tu, EF-Ts and the N-terminal half of S1, which is capable of initiating Qß RNA replication. Structural and biochemical studies revealed that the two N-terminal OB-folds of S1 anchor S1 onto the ß-subunit, and the third OB-fold is mobile and protrudes beyond the surface of the ß-subunit. The third OB-fold mainly interacts with a specific RNA fragment derived from the internal region of Qß RNA, and its RNA-binding ability is required for replication initiation of Qß RNA. Thus, the third mobile OB-fold of S1, which is spatially anchored near the surface of the ß-subunit, primarily recruits the Qß RNA toward the ß-subunit, leading to the specific and efficient replication initiation of Qß RNA, and S1 functions as a replication initiation factor, beyond its established function 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.

Ribosomal protein S1 functions as a termination factor in RNA synthesis by Qß phage replicase.

S1 is the largest ribosomal protein, and is vitally important for the cell. S1 is also a subunit of Qß replicase, the RNA-directed RNA polymerase of bacteriophage Qß. In both protein and RNA syntheses, S1 is commonly believed to bind to a template RNA at the initiation step, and not to be involved in later events. Here, we show that in Qß replicase-mediated RNA synthesis, S1 functions at the termination step by promoting release of the product strand in a single-stranded form. This function is fulfilled by the N-terminal fragment comprising the first two S1 domains. The results suggest that S1 might also have a role other than mRNA binding in the ribosome.

Molecular basis for RNA polymerization by Qß replicase.

Core Qß replicase comprises the Qß virus-encoded RNA-dependent RNA polymerase (ß-subunit) and the host Escherichia coli translational elongation factors EF-Tu and EF-Ts. The functions of the host proteins in the viral replicase are not clear. Structural analyses of RNA polymerization by core Qß replicase reveal that at the initiation stage, the 3'-adenine of the template RNA provides a stable platform for de novo initiation. EF-Tu in Qß replicase forms a template exit channel with the ß-subunit. At the elongation stages, the C-terminal region of the ß-subunit, assisted by EF-Tu, splits the temporarily double-stranded RNA between the template and nascent RNAs before translocation of the single-stranded template RNA into the exit channel. Therefore, EF-Tu in Qß replicase modulates RNA elongation processes in a distinct manner from its established function in protein synthesis.

[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.