A temperature-sensitive mutation affecting the mammalian 60 S ribosome

A temperature-sensitive Chinese hamster cell mutant, ts14, is unable to synthesize protein in tissue culture at 39 degrees. That mutant's protein biosynthetic machinery has been characterized in cell-free, biologically active extracts. Similar to the mutant's phenotype in tissue culture, ts14 extracts cease protein synthesis in vitro within 15 min at 40 degrees. In contrast, at 25 degrees both ts14 and wild type extracts synthesize protein for more than 2 hours. Fractionation of mutant extracts and complementation with comparable wild type preparations indicate that ts14 possesses a thermolabile component associated with its polyribosomes. In preparation of ts14 ribosomes that are free of mRNA and bound protein factors, the defective factor is complemented functionally only by 60 S ribosomal subunits prepared from the wild type parent. Sedimentation analyses in sucrose gradients demonstrate that ts14's mutation specifically affects stability of the mutant's 60 S ribosome. Treatment with high ionic strength buffers preferentially disrupts the mutant's 60 S ribosomal subunit and results in preparations of mutant ribosomes that contain biologically active 40 S subunits only. These studies demonstrate the applicability of a genetic approach to analyzing structure-function relationships in the eukaryotic ribosome.