title: Genetically engineered active Qbeta replicase in rabbit reticulocyte cell-free system: a fusion protein of EF-Tu and EF-Ts is functional as the subunit of Qbeta replicase.
abstract: Qbeta replicase functioning in Escherichia coli is an RNA-dependent RNA polymerase composed of one phage-coded subunit and three host-coded proteins: ribosomal protein S1, and protein elongation factors EF-Tu and EF-Ts. Qbeta replicase lacking ribosomal protein S1 (alpha-less replicase) is capable of replicating some small RNAs. We attempted to create functional alpha-less replicase by co-expression of the mRNAs that code for the subunits of alpha-less replicase in a rabbit reticulocyte cell-free translation system. Replicase activity, however, could not be detected when both EF-Tu and EF-Ts were co-expressed with the phage-coded subunit. On the other hand, active alpha-less replicase was obtained when an EF-Ts-EF-Tu fusion protein was co-expressed with the phage-coded subunit. Consequently, we succeeded in generating genetically engineered active alpha-less Qbeta replicase which functions in a eukaryotic cell-free system.
authors.affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
authors.forename: Hajime
authors.initials: H
authors.lastname: Fukano
authors.forename: Zako
authors.initials: Z
authors.lastname: Tamotsu
authors.forename: Suzuki
authors.initials: S
authors.lastname: Eiji
authors.forename: Watanabe
authors.initials: W
authors.lastname: Kimitsuna
authors.forename: Nagamune
authors.initials: N
authors.lastname: Teruyuki