Assembly map of the large subunit (50S) of Escherichia coli ribosomes.

Abstract

Stoichiometric amounts of ribosomal proteins and RNA derived from the 50S subunit reconstitute to fully active particles under the conditions of a two-step incubation procedure. After the first incubation, all components are found in a particle that is activated in the second incubation [Dohme, F. & Nierhaus, K. H. (1976) J. Mol. Biol. 107, 585-599]. Here we describe the assembly dependences of the ribosomal components in the first incubation. Assembly dependence is the requirements of one protein that, before it binds, another must be first built into the ribosome. After incubation of 23S RNA and the proteins under observation, the mixture was subjected to sucrose gradient analysis. The RNA-protein complex was precipitated with trichloroacetic acid and the proteins were identified by NaDodSO4 gel electrophoresis. The assembly dependences of 26 proteins could be elucidated. In a second series of experiments, the incorporation of 3H-labeled 5S RNA in the 23S-protein complex was analyzed. It was found that L5, L15, and L18 are absolutely required for 5S RNA incorporation. In addition, two of the three proteins L2, L3, and L4 are needed, in excellent agreement with the protein dependences. The data are summarized in an assembly map. Comparison with other data shows a structural domain at the 5' end of 23S RNA around protein L20 combining all proteins essential in the early assembly. All the proteins essential for the reconstitution of the peptidyltransferase protein form a skeleton of strong assembly dependences. Finally, L proteins whose genes are present in large transcriptional units on the chromosome depend on each other during assembly.