Functional organization of the large ribosomal subunit of Bacillus stearothermophilus

Abstract

Bacillus stearothermophilus 50 S ribosomal subunits active in polyphenylalanine (polyPhe) synthesis were reconstituted from a mixture of purified proteins and RNA. Proteins were omitted one at a time, and the resulting particles were examined by sucrose gradient sedimentation and assayed for polyPhe synthesis, peptidyltransferase activity, and in some cases binding of elongation factor EF-G and GTP, and association with a (20 S . Phe-tRNA . poly(U)) complex. Based on their effect on polyPhe synthesis and peptidyltransferase activity, the proteins were grouped into four functional categories. The set of proteins most strongly required for peptidyltransferase activity, which must include the protein or proteins most directly involved in the active center, consists of proteins (probable Escherichia coli homologs in parentheses) B-L3 (E-L2), B-L4 (E-L4), B-L5 (E-L5), B-L6 (E-L3 or E-L6), B-L18 (E-L14), B-L20b (E-L16), and B-L25 (E-L20). Several proteins affected both polyPhe synthesis and peptidyltransferase activity more weakly. Only four proteins were required for polyPhe synthesis but not for peptidyltransferase activity, B-L2 (E-L1), B-L8 (E-L10), B-L13 (E-L7/L12), and B-L11(E-L11). The results indicate that the peptidyltransferase center is tightly integrated into the cooperative body of the 50 S subunit and that the (B-L8 . B-L13) complex is relatively independent of this cooperative domain.