Properties of the ribosomes of bacterial mutants resistant to thiostrepton.

A protein required for the binding of thiostrepton to ribosomes from Bacillus megaterium has been purified and shown to be serologically homologous with Esoherioiiia coli ribosomal protein L11. Addition of this B. megaterium protein (designated protein BM-Lll) to 23S rRNA is sufficient to create a high-affinity thiostrepton binding site present on neither protein nor RNA alone. Examination in vitro of four independently-arising thiostrepton- resistant mutants of B. megaterium revealed that drug resistance was in each case a property of the ribosomes, which were unable to bind [35s] thiostrepton with high affinity. Addition of protein EM-Lll to such ribosomes restored their ability to bind the drug tightly. Weak interaction of [35s] thiostrepton with ribosomes form one of the mutants (mutant MJl) was detected using equilibrium dialysis (Kd = 2 x 10-7 M). Use of two-dimensional polyacrylamide gel electrophoresis suggested that ribosomes from the mutant strains lacked protein BM-L11, a conclusion verified by the results of immunological analyses. Such ribosomes showed only partial activity in protein synthesis in vitro when compared with ribosomes from the wild-type, but re-addition of protein BM-L11 restored the former to wild-type levels of activity. Examination of individual steps involved in polypeptide chain elongation demonstrated the ribosomes from mutant MJ1 were impaired in their ability to hydrolyze GTP in the presence of EF G. Reconstitution of the ribosomes with either B. megaterium protein BM-Lll or E. coli protein L11 reversed this deficiency, indicating a role for protein BM-Lll (and, by inference, E. coli protein L11) in EF G-dependent GTP hydrolysis and showing a functional relationship between the two proteins. However, protein BM-L11 is evidently not essential for ribosomal function. Unlike the parental B. megaterium strain, mutant MJ1 is phenotypically relaxed. This can be directly attributed to lack of protein BM-L11 from the ribosomes. Thus ribosomes from mutant MJ1 do not synthesise (p)ppGpp in vivo during isoleucine starvation and can only produce these regulatory nucleotides in vitro when supplemented with protein BM-L11.