2Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France; E-mail: email@example.com
3INSERM, U964, Illkirch, F-67400, France
* To whom correspondence should be addressed.
Received March 13, 2010; Revision received April 8, 2010
Qβ replicase is a protein complex responsible for the replication of the genomic RNA of bacteriophage Qβ. In addition to the phage-encoded catalytic β 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 Qβ 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 β 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 Qβ replicase and the mechanism of recognition of its templates that can replicate in vitro at a record rate.
KEY WORDS: RNA replication, RNA-dependent RNA polymerase, ribosomal protein S1, elongation factors EF-Tu and EF-Ts, two plasmid expression system, protein crystallization, structure stability