Structure-function relationships in the ribosomal protein L12 in the archaeon Sulfolobus acidocaldarius

A series of mutant L12 ribosomal proteins was prepared by site-directed mut ations in the L12 protein gene of the archaeon Sulfolobus acidocaldarius. T he mutant protein genes were overexpressed in Escherichia coli, and the pro ducts purified and incorporated into ribosomal cores which had been ethanol extracted to remove wild-type L12 protein. Measurements were made to deter mine if the mutation affected the binding of the L12 protein to the ribosom e core or affected the translational activity of the resulting ribosome. Ch anging tyrosine [3] or tyrosine [5], conserved in all archaea and present i n all eukarya in positions [3] and [7], to phenylalanine had no effect on b inding or translational activity while changes to glycine significantly red uced binding and translational activity. Changing the single arginine [37] residue, conserved in almost all archaeal and eukaryal L12 proteins, to lys ine, glutamic acid, glutamine, or glycine had no effect on binding to the c ore and had little or no significant effect on translational activity. The same was true when lysine [39], conserved in all archaeal L12 proteins, was changed to arginine, glutamic acid, glutamine, or glycine. Changing phenyl alanine [104], the penultimate amino acid at the C-terminal end, which is c onserved in all archaeal and eukaryal L12 proteins, to tyrosine or glycine had no effect on binding but lowered the translational activity by 60 and 7 5%, respectively, suggesting that this amino acid plays an important role i n translation. Deletion of the highly charged region in the C-terminal doma in, which is present in all archaeal and eukaryal L12 proteins, decreased t ransitional activity by 50%, suggesting this region is also involved in fac tor interactions. (C) 1999 Academic Press.