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.

Transient compartmentalization of RNA replicators prevents extinction due to parasites.

The appearance of molecular replicators (molecules that can be copied) was probably a critical step in the origin of life. However, parasitic replicators would take over and would have prevented life from taking off unless the replicators were compartmentalized in reproducing protocells. Paradoxically, control of protocell reproduction would seem to require evolved replicators. We show here that a simpler population structure, based on cycles of transient compartmentalization (TC) and mixing of RNA replicators, is sufficient to prevent takeover by parasitic mutants. TC tends to select for ensembles of replicators that replicate at a similar rate, including a diversity of parasites that could serve as a source of opportunistic functionality. Thus, TC in natural, abiological compartments could have allowed life to take hold.

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.

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.

Kinetic model of double-stranded RNA formation during long RNA replication by Qß replicase.

Qß replicase is an RNA-dependent RNA polymerase, which synthesizes the complementary RNA using a single-stranded RNA as a template. The formation of non-replicable double-stranded RNA (dsRNA) by hybridization between newly synthesized RNA and the template RNA hinders the broader application of Qß replicase. Here, we developed a kinetic model of Qß RNA replication consisting of two reaction pathways of dsRNA formation, which quantitatively explains the dynamics of dsRNA formation of three template RNAs. We also found that part of the Qß phage genomic RNA sequence including the central hairpin loop significantly decreases the rate of dsRNA formation.

Mechanism for template-independent terminal adenylation activity of Qß replicase.

The genomic RNA of Qß virus is replicated by Qß replicase, a template-dependent RNA polymerase complex. Qß replicase has an intrinsic template-independent RNA 3'-adenylation activity, which is required for efficient viral RNA amplification in the host cells. However, the mechanism of the template-independent 3'-adenylation of RNAs by Qß replicase has remained elusive. We determined the structure of a complex that includes Qß replicase, a template RNA, a growing RNA complementary to the template RNA, and ATP. The structure represents the terminal stage of RNA polymerization and reveals that the shape and size of the nucleotide-binding pocket becomes available for ATP accommodation after the 3'-penultimate template-dependent C-addition. The stacking interaction between the ATP and the neighboring Watson-Crick base pair, between the 5'-G in the template and the 3'-C in the growing RNA, contributes to the nucleotide specificity. Thus, the template for the template-independent 3'-adenylation by Qß replicase is the RNA and protein ribonucleoprotein complex.

[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.

Isolation and crystallization of a chimeric Qß replicase containing Thermus thermophilus EF-Ts.

Qß replicase is a protein complex responsible for the replication of the genomic RNA of bacteriophage Qß. In addition to the phage-encoded catalytic ß subunit, it recruits three proteins from the host Escherichia coli cell: elongation factors EF-Tu and EF-Ts and ribosomal protein S1. We prepared a chimeric Qß replicase in which the E. coli EF-Ts is replaced with EF-Ts from Thermus thermophilus. The chimeric protein is produced in E. coli cells during coexpression of the genes encoding the ß subunit and thermophilic EF-Ts. The developed isolation procedure yields a substantially homogeneous preparation of the chimeric replicase. Unlike the wild-type enzyme, the S1-less chimeric replicase could be crystallized. This result facilitates studies on the structure of Qß replicase and the mechanism of recognition of its templates that can replicate in vitro at a record rate.

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.

Compartmentalization in a water-in-oil emulsion repressed the spontaneous amplification of RNA by Q beta replicase.

During RNA replication mediated by Qbeta replicase, self-replicating RNAs (RQ RNAs) are amplified without the addition of template RNA. This undesired amplification makes the study of target RNA replication difficult, especially for long RNA such as genomic RNA of Qbeta phage. This perhaps is one of the reasons why the precise rate of genomic RNA replication in the presence of host factor Hfq has not been reported in vitro. Here, we report a method to repress RQ RNA amplification by compartmentalization of the reaction using a water-in-oil emulsion but maintaining the activity of Qbeta replicase. This method allowed us to amplify the phage Qbeta genome RNA exponentially without detectable amplification of RQ RNA. Furthermore, we found that the rate constant of genome RNA replication in the exponential phase at the optimum Hfq concentration was approximately 4.6 times larger than that of a previous report, close to in vivo data. This result indicates that the replication rate in vivo is largely explained by the presence of Hfq. This easy method paves the way for the study of genomic RNA replication without special care for the undesired RQ RNA amplification.

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.

[Scientific and practical applications of molecular colonies].

Reviewed are the history of invention of the molecular colony technique, also known under name "polony technology", applications of this method to studies of reactions between single RNA molecules, ultrasensitive diagnostics, gene cloning and screening in vitro, and also concepts on the origin of life that consider molecular colonies as a prototype of living organisms.

Kinetic analysis of the entire RNA amplification process by Qbeta replicase.

The kinetics of the RNA replication reaction by Qbeta replicase were investigated. Qbeta replicase is an RNA-dependent RNA polymerase responsible for replicating the RNA genome of coliphage Qbeta and plays a key role in the life cycle of the Qbeta phage. Although the RNA replication reaction using this enzyme has long been studied, a kinetic model that can describe the entire RNA amplification process has yet to be determined. In this study, we propose a kinetic model that is able to account for the entire RNA amplification process. The key to our proposed kinetic model is the consideration of nonproductive binding (i.e. binding of an enzyme to the RNA where the enzyme cannot initiate the reaction). By considering nonproductive binding and the notable enzyme inactivation we observed, the previous observations that remained unresolved could also be explained. Moreover, based on the kinetic model and the experimental results, we determined rate and equilibrium constants using template RNAs of various lengths. The proposed model and the obtained constants provide important information both for understanding the basis of Qbeta phage amplification and the applications using Qbeta replicase.

In vitro improvement of a shark IgNAR antibody by Qbeta replicase mutation and ribosome display mimics in vivo affinity maturation.

We have employed a novel mutagenesis system, which utilizes an error-prone RNA dependent RNA polymerase from Qbeta bacteriophage, to create a diverse library of single domain antibody fragments based on the shark IgNAR antibody isotype. Coupling of these randomly mutated mRNA templates directly to the translating ribosome allowed in vitro selection of affinity matured variants showing enhanced binding to target, the apical membrane antigen 1 (AMA1) from Plasmodium falciparum. One mutation mapping to the IgNAR CDR1 loop was not readily additive to other changes, a result explained by structural analysis of aromatic interactions linking the CDR1, CDR3, and Ig framework regions. This combination appeared also to be counter-selected in experiments, suggesting that in vitro affinity maturation is additionally capable of discriminating against incorrectly produced protein variants. Interestingly, a further mutation was directed to a position in the IgNAR heavy loop 4 which is also specifically targeted during the in vivo shark response to antigen, providing a correlation between natural processes and laboratory-based affinity maturation systems.