RIBOSOMAL-PROTEIN S15 FROM THERMUS-THERMOPHILUS - CLONING, SEQUENCING, OVEREXPRESSION OF THE GENE AND RNA-BINDING PROPERTIES OF THE PROTEIN

A 6-kb DNA fragment from an extreme thermophile, Thermus thermophilus, carrying the genes for cytochrome oxidase ba(3) subunit I(cbaA) and t he ribosomal protein S15 (rpsO) was cloned into Escherichia coli. The gene rpsO was sequenced. The deduced amino acid sequence exhibits 59% identity to the corresponding protein from E. coli. Expression of rpsO in E. coli requires the use of a-fully repressed inducible promoter b ecause S15 from T. thermophilus is toxic for E. coli cells. When purif ied without denaturation from either overproducing E. coli strain or f rom T. thermophilus ribosomes, the S15 protein is stable and binds a c loned T. thermophilus 16S rRNA fragment (nucleotides 559-753), with lo w identical dissociation constants (2.5 nM), thus demonstrating that t he thermophilic protein folds correctly in a mesophilic bacterium. The rRNA fragment bound corresponds in position and structure to the 16S rRNA fragment of E. coli. A similar high affinity was also found for t he binding of S15 from T. thermophilus or E. coli to the corresponding E. coli 16S rRNA fragment, whereas a slightly lower affinity was obse rved in binding experiments between E. coli S15 and T. thermophilus 16 S rRNA fragment. These results suggest that S15 from T. thermophilus r ecognizes similar determinants in both rRNA fragments. Competition exp eriments support this conclusion.