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 requirement 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 IA 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 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.The assembly of the small subunit (30S) of Escherichia coli ribosomes has been studied in detail by the reconstitution technique, and much information has accumulated about the precise assembly dependences of the 30S components (30S assembly map; for review, see ref. MATERIALS AND METHODS 70S ribosomes and 23S and 5S RNAs were prepared from E. coli cells as described (6). For the isolation of 5S [3H]RNA, E. coli cells (MRE600) were grown in 2 liters of minimal medium (7). When the culture reached an OD650nm of 0.06 units/ml, 300 nmol of [5,6-3H]uridine (specific activity, 40 Ci/mmol; 1 Ci = 3.7 x 1010 becquerels) was added, and the cells (5 g) were harvested one generation later. The isolation procedure of 5S [3H]RNA followed ref. 6. However, to reduce radiolysis, the material was processed as quickly as possible and not frozen until the labeled RNA was isolated. The specific activity was 1,000,000 cpm/A2nm unit. The isolation of highly purified ribosomal proteins followed ref. 8.Standard Assembly Mapping Experiments. These experiments were as reported (5) with slight modifications. Five A260nm units of23S RNA and 10 equivalent units (where 1 equivalent unit is the amount of protein on 1 Aaw . unit of 50S subunits) of proteins were incubated for 20 min at 44°C in 200 ;LI of 20 mM Tris-HCl, pH 7.5/4 mM Mg(OAc)2/0.2 mM EDTA/ 400 mM NH4CV/4 mM 2-mercaptoethanol (i.e., the conditions...

No abstract

Ribosomes carrying either a normally methylated or an undermethylated L11, respectively, were tested with respect to the stringency reaction in the presence of crude stringent factor. Systems with either kind of ribosomes synthesize ppGpp with the same efficiency. The ppGpp synthesis in presence of ribosomes with undermethylated L11 depends also on stringent factor, mRNA and deacylated tRNA whereas aminoacyl-tRNA and peptidyl-analogous tRNA show no effect. Thus, the absence of methylation in L11 does not influence the stringency reaction.

No abstract