Qβ Replicase Research Articles

Probing the legitimate initiation of RNA synthesis by Qβ 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 Qβ 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, Qβ replicase binds the 5'-triphosphate of the initiating nucleotide GTP, and this binding is needed for the replication of legitimate templates.

Construction of a mini-RNA replicon in Escherichia coli.

How the ribonucleic acid (RNA) world transited to the deoxyribonucleic acid (DNA) world has remained controversial in evolutionary biology. At a certain time point in the transition from the RNA world to the DNA world, 'RNA replicons', in which RNAs produce proteins to replicate their coding RNA, and 'DNA replicons', in which DNAs produce RNA to synthesize proteins that replicate their coding DNA, can be assumed to coexist. The coexistent state of RNA replicons and DNA replicons is desired for experimental approaches to determine how the DNA world overtook the RNA world. We constructed a mini-RNA replicon in Escherichia coli. This mini-RNA replicon encoded the β subunit, one of the subunits of the Qβ replicase derived from the positive-sense single-stranded Qβ RNA phage and is replicated by the replicase in E. coli. To maintain the mini-RNA replicon persistently in E. coli cells, we employed a system of α complementation of LacZ that was dependent on the Qβ replicase, allowing the cells carrying the RNA replicon to grow in the lactose minimal medium selectively. The coexistent state of the mini-RNA replicon and DNA replicon (E. coli genome) was successively synthesized. The coexistent state can be used as a starting system to experimentally demonstrate the transition from the RNA-protein world to the DNA world, which will contribute to progress in the research field of the origin of life.

Involvement of RNA chaperone hfq in the regulation of antibiotic resistance and virulence in Shigella sonnei

The host factor for RNA phage Qβ replicase (Hfq) is a crucial post-transcriptional regulator in many bacterial pathogens, facilitating the interaction between small non-coding RNAs (sRNAs) and their target mRNAs. Studies have suggested that Hfq plays a role in antibiotic resistance and virulence in bacteria, although its functions in Shigella are not fully understood. In this study, we investigated the functional roles of Hfq in Shigella sonnei (S. sonnei) by constructing an hfq deletion mutant. Our phenotypic assays showed that the hfq deletion mutant was more sensitivity to antibiotics and had impaired virulence. Transcriptome analyses supported the results concerning the phenotype of the hfq mutant and showed that differentially expressed genes were mainly enriched in the KEGG pathways two-component system, ABC transporters, ribosome, and Escherichia coli biofilm formation. Additionally, we predicted eleven novel Hfq-dependent sRNAs, which were potentially involved in the regulation of antibiotic resistance and/or virulence in S. sonnei. Our findings suggest that Hfq plays a post-transcriptional role in regulating antibiotic resistance and virulence in S. sonnei, and could provide a basis for future studies on Hfq-sRNA-mRNA regulatory networks in this important pathogen.

Slippage at the initiation of RNA synthesis by Qβ 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, Qβ replicase, the RNA-directed RNA polymerase of bacteriophage Qβ, 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 Qβ 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.

Relaxed Substrate Specificity in Qβ Replicase through Long-Term In Vitro Evolution

A change from RNA- to DNA-based genetic systems is hypothesized as a major transition in the evolution of early life forms. One of the possible requirements for this transition is a change in the substrate specificity of the replication enzyme. It is largely unknown how such changes would have occurred during early evolutionary history. In this study, we present evidence that an RNA replication enzyme that has evolved in the absence of deoxyribonucleotide triphosphates (dNTPs) relaxes its substrate specificity and incorporates labeled dNTPs. This result implies that ancient replication enzymes, which probably evolved in the absence of dNTPs, could have incorporated dNTPs to synthesize DNA soon after dNTPs became available. The transition from RNA to DNA, therefore, might have been easier than previously thought.

Corrigendum: A Fusion Method to Develop an Expanded Artificial Genomic RNA Replicable by Qβ Replicase

One or two additional mutations, which were not included in the analysis, were found in seven RNAs (G2168U in #5, G284U and C2462A in #7, C1253A and C1879A in #8, C2016A in #9, C1327U in #11, G535U in #12, G816U and G1147U in #13, and C1094A and C1140A in #22). These mutations slightly change Figure 1 C and D, as shown below, and the correlation coefficients from −0.092 to 0.043 and from −0.51 to −0.64, respectively. These changes do not affect the conclusion. Replication of fused RNAs with purified replicase. C) Relationship between the previous parameter, GC number in loops, and ssRNA synthesis. The correlation efficient is 0.043. D) Relationship between the new parameter, the unreplicable value, and ssRNA synthesis. The correlation efficient is −0.64. The authors apologize for this oversight.

Structural transition of replicable RNAs during in vitro evolution with Qβ 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 Qβ 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 Qβ 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 Qβ replicase, useful for more complex ssRNA replication systems.

Unsolved Puzzles of Qβ Replicase

Unsolved Puzzles of Qβ Replicase

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

Unsolved Puzzles of Qβ Replicase

Qβ phage replicase has been the first RNA-directed RNA polymerase purified to homogeneity and intensively studied in vitro. In the mid-sixties, papers on Qβ 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 Qβ replicase did not help much. Certainly, there remains a lot to discover in the replication of Qβ phage, one of the smallest viruses known.

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

Qβ replicase (RNA-directed RNA polymerase of bacteriophage Qβ) 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 Qβ replicase, discovering the ability of RNA molecules for spontaneous recombination, revealing the unusual mechanism Qβ 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, Qβ replicase has not yet revealed all its secrets, and its studies promise further interesting findings.

Mycoplasma pneumoniae: A significant but underrated pathogen in paediatric community-acquired lower respiratory tract infections.

Lower respiratory tract infections are considered a common cause responsible for morbidity and mortality among children, and Mycoplasma pneumoniae is identified to be responsible for up to 40 per cent of community-acquired pneumonia in children greater than five years of age. Extrapulmonary manifestations have been reported either due to spread of infection or autoimmune mechanisms. Infection by M. pneumoniae has high incidence and clinical importance but is still an underrated disease. Most widely used serologic methods are enzyme immunoassays for detection of immunoglobulin M (IgM), IgG and IgA antibodies to M. pneumoniae, though other methods such as particle agglutination assays and immunofluorescence methods are also used. Detection of M. pneumoniae by nucleic acid amplification techniques provides fast, sensitive and specific results. Utilization of polymerase chain reaction (PCR) has improved the diagnosis of M. pneumoniae infections. Besides PCR, other alternative amplification techniques include (i) nucleic acid sequence-based amplification, (ii) Qβ replicase amplification, (iii) strand displacement amplification, (iv) transcription-mediated amplification, and (v) ligase chain reaction. Macrolides are used as the first-line treatment in childhood for M. pneumoniae infections; however, emergence of macrolide-resistant M. pneumoniae is a cause of concern. Development of a safe vaccine is important that gives protective immunity and would be a major step in reducing M. pneumoniae infections.

The Contribution of Ribosomal Protein S1 to the Structure and Function of Qβ Replicase

The high resolution crystal structure of bacterial ribosome was determined more than 10 years ago; however, it contains no information on the structure of the largest ribosomal protein, S1. This unusual protein comprises six flexibly linked domains; therefore, it lacks a fixed structure and this prevents the formation of crystals. Besides being a component of the ribosome, protein S1 also serves as one of the four subunits of Q replicase, the RNA-directed RNA polymerase of bacteriophage Q. In each case, the role of this RNA-binding protein has been thought to consist in holding the template close to the active site of the enzyme. In recent years, a breakthrough was made in studies of protein S1 within Q replicase. This includes the discovery of its paradoxical ability to displace RNA from the replicase complex and determining the crystal structure of its fragment capable of performing this function. The new findings call for a re-examination of the contribution of protein S1 to the structure and function of the ribosome.

Вклад рибосомного белка S1 в структуру и функцию Qβ-репликазы

The high resolution crystal structure of bacterial ribosome was determined more than 10 years ago; however, it contains no information on the structure of the largest ribosomal protein, S1. This unusual protein comprises six flexibly linked domains; therefore, it lacks a fixed structure and this prevents the formation of crystals. Besides being a component of the ribosome, protein S1 also serves as one of the four subunits of Qβ replicase, the RNA-directed RNA polymerase of bacteriophage Qβ. In each case, the role of this RNA-binding protein has been thought to consist in holding the template close to the active site of the enzyme. In recent years, a breakthrough was made in studies of protein S1 within Qβ replicase. This includes the discovery of its paradoxical ability to displace RNA from the replicase complex and determining the crystal structure of its fragment capable of performing this function. The new findings call for a re-examination of the contribution of protein S1 to the structure and function of the ribosome.

Вклад рибосомного белка S1 в структуру и функцию Qβ-репликазы

Вклад рибосомного белка S1 в структуру и функцию Qβ-репликазы

Combinatorial selection for replicable RNA by Qβ 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 Qβ 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 Qβ 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 β-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 Qβ 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 Qβ 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 Qβ replicase, which was translated from the RNA in giant liposomes encapsulating the cell-free translation system. A reporter RNA encoding the antisense strand of β-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 Qβ replication.

Upon infection of Escherichia coli by bacteriophage Qβ, the virus-encoded β-subunit recruits host translation elongation factors EF-Tu and EF-Ts and ribosomal protein S1 to form the Qβ replicase holoenzyme complex, which is responsible for amplifying the Qβ (+)-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 β-subunit and the first two oligonucleotide-oligosaccharide-binding domains of S1 (OB1–2) in the recognition of Qβ (+)-RNA by the Qβ replicase complex. We show how three basic residues of the β 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 Qβ 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 β-subunit and protein S1 cooperatively bind the (+)-stranded Qβ genome during replication initiation and provide a foundation for understanding template discrimination during replication initiation.

Replication of partial double-stranded RNAs by Qβ replicase

Qβ replicase, an RNA-dependent RNA polymerase of bacteriophage Qβ, 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 Qβ 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 Qβ replicase. We found that Qβ 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 Qβ replicase.

A design principle for a single-stranded RNA genome that replicates with less double-strand formation.

Single-stranded RNA (ssRNA) is the simplest form of genetic molecule and constitutes the genome in some viruses and presumably in primitive life-forms. However, an innate and unsolved problem regarding the ssRNA genome is formation of inactive double-stranded RNA (dsRNA) during replication. Here, we addressed this problem by focusing on the secondary structure. We systematically designed RNAs with various structures and observed dsRNA formation during replication using an RNA replicase (Qβ replicase). From the results, we extracted a simple rule regarding ssRNA genome replication with less dsRNA formation (less GC number in loops) and then designed an artificial RNA that encodes a domain of the β-galactosidase gene based on this rule. We also obtained evidence that this rule governs the natural genomes of all bacterial and most fungal viruses presently known. This study revealed one of the structural design principles of an ssRNA genome that replicates continuously with less dsRNA formation.