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.

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 Qbeta replicase genetically fusing essential subunits EF-Ts and EF-Tu with beta-subunit.

Qbeta replicase, an RNA-dependent RNA polymerase of RNA coliphage Qbeta, is a heterotetramer composed of a phage-encoded beta-subunit and three host-encoded proteins: the ribosomal protein S1 (alpha-subunit), EF-Tu, and EF-Ts. Several purification methods for Qbeta replicase were described previously. However, in our efforts to improve the production of Qbeta replicase, a substantial amount of the beta-subunit overproduced in Escherichia coli cells was found as insoluble aggregates. In this paper, we describe two kinds of method of producing Qbeta replicase. In one kind, both EF-Tu and EF-Ts subunits were expressed with the beta-subunit, and in the other kind, the beta-subunit was genetically fused with EF-Tu and EF-Ts. The fused protein, a single-chain alpha-less Qbeta replicase, was mostly found in the soluble fraction and could be readily purified. These results pave the way for the large-scale production of the highly purified form of this enzyme.

Facile production and rapid purification of functional recombinant Qß replicase heterotetramer complex.

We describe an improved method for the production of recombinant Qß replicase heterotetramer. The successful expression of the soluble Qß RNA polymerase complex depends on the EF-Ts and EF-Tu subunits being co-expressed prior to ß-subunit expression. Efficient co-expression requires two different inducible operons to co-ordinate the expression of the heterotrimer. The complete heterotetramer enzyme complex is achieved by production of the recombinant S1-subunit of Qß replicase in a separate host. This approach represents a facile way for producing and purifying large amounts of soluble and active recombinant Qß replicase tetramer without the necessity of a His-tag for purification.

Q beta replicase containing wild type and mutant tufA and tufB gene.

The protein synthesis elongation factor EF-Tu, complexed with EF-Ts, forms part of Q beta RNA replicase. In an effort to determine its function in the RNA synthesis reaction, we have developed procedures which allow us to replace the endogenous EF-Tu in purified Q beta replicase with EF-Tu from a variety of sources. In this communication we report purification of EF-Tu from strains containing (a) a wild type tufA gene only, (b) a kirromycin-resistant mutant tufA gene only, and (c) a kirromycin-resistant mutant tufA gene and a mutant tufB gene which codes for EF-Tu that does not bind ribosomes. When each of these EF-Tu preparations is inserted in Q beta replicase, the wild type tufA gene product and and the tufB gene product function appearently normally, but the kirromycin-resistant tufA gene product causes the formation of an altered enzyme. The Q beta replicase containing kirromycin-resistant EF-Tu is unstable; it is rapidly inactivated in the reaction mixture, even at temperatures as low as 20 degrees C. This property results in an apparent increase in template specificity; while wild type Q beta replicase will transcribe poly(C) and other synthetic RNA species, the mutant enzyme will do so only in the presence of Mn2+, which reduces template specificity. The kirromycin-resistant Q beta replicase will also transcribe Q beta RNA. The results imply that EF-Tu is involved in maintenance of enzyme structure, which, in turn, is implicated in template specificity.