Assaying RNA chaperone activity in vivo in bacteria using a ribozyme folding trap.
Here, we report an assay to evaluate the intracellular RNA chaperone activity of a protein of interest in vivo in bacterial cells. The method is based on self-splicing of the group I intron, which is located in the thymidylate synthase (td) gene of phage T4. A previously described td mutant (tdSH1) has significantly impaired splicing due to formation of splicing-incompetent alternative structures. In this procedure, overexpression of RNA chaperones in the presence of the td mutant SH1 is used to evaluate whether the putative RNA chaperone is able to rescue the incorrectly folded group I intron. The ability of the RNA chaperone to assist during folding is measured indirectly by assessing the difference between the splicing efficiencies of the td mutant in the absence and in the presence of the RNA chaperone. This procedure can be completed in 5-6 d, not including the time needed to clone the putative RNA chaperone.
Prenninger
Silvia
S
Max F. Perutz Laboratories, University of Vienna, Dept. of Biochemistry, Dr. Bohrgasse 9/5, A-1030 Vienna, Austria.
Schroeder
Renée
R
Semrad
Katharina
K
D017122:Bacteriophage T4 / Q000235:genetics; D001681:Biological Assay / Q000379:methods*; D017931:DNA Primers; D004926:Escherichia coli; D005822:Genetic Vectors / Q000235:genetics; D007438:Introns / Q000235:genetics; D018832:Molecular Chaperones / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D012313:RNA / Q000378:metabolism*; D016337:RNA, Catalytic; D016601:RNA-Binding Proteins / Q000378:metabolism*; D013940:Thymidylate Synthase / Q000235:genetics
D017931:DNA Primers; D018832:Molecular Chaperones; D016337:RNA, Catalytic; D016601:RNA-Binding Proteins; D012313:RNA; D013940:Thymidylate Synthase