Efficient reconstitution of functional Escherichia coli 30S ribosomal subunits from a complete set of recombinant small subunit ribosomal proteins

Previous Studies have shown that the 30S ribosomal subunit of Escherichia c oli can be reconstituted in vitro from individually purified ribosomal prot eins and 16S ribosomal RNA, which were isolated from natural 30S subunits. We have developed a 30S subunit reconstitution system that uses only recomb inant ribosomal protein components. The genes encoding E. coli ribosomal pr oteins S2-S21 were cloned,and all twenty of the individual proteins were ov erexpressed and purified. Reconstitution, following standard procedures, us ing the complete set of recombinant proteins and purified 16S ribosomal RNA is highly inefficient. Efficient reconstitution of 30S subunits using thes e components requires sequential addition of proteins, following either the 30S subunit assembly map (Mizushima & Noura, 1970, Nature 226:1214-1218; H eld et al., 1974, J Biol Chem 249:3103-3111) or following the order of prot ein assembly predicted from in vitro assembly kinetics (Powers et al., 1993 ; J Mol Biol 232:362-374)In the first procedure, the proteins were divided into three groups, Group I (S4, S7, S8, S15, S17, and S20), Group II (S5, S 6, S9, S11, S12, S13, S16, S18, and S19), and Group III (S2, S3, S10, S14, and S21), which were sequentially added to 16S rRNA with a 20 min incubatio n at 42 degrees C following the addition of each group. In the second proce dure; the proteins were divided into Group I (S4, S6, S11, S15, S16, S17, S 18, and S20), Group ll:(S7, S8, S9, S13, and S19), Group II' (S5 and S12) a nd Group III (S2, S3, S10, S14, and S21). Similarly efficient reconstitutio n is observed whether the proteins are grouped according to the assembly ma p or according to the results of in vitro 30S subunit assembly kinetics. Although reconstitution of 30S subunits using the recombinant proteins is s lightly less efficient than reconstitution using a mixture of total protein s isolated from 30S subunits, it is much more efficient than reconstitution using proteins that were individually isolated from ribosomes. Particles r econstituted from the recombinant proteins sediment at 30S in sucrose gradi ents, bind tRNA in a template-dependent manner, and associate with 50S subu nits to form 70S ribosomes that are active in poly(U)-directed polyphenylal anine synthesis. Both the protein composition and the dimethyl sulfate modi fication pattern of 16S ribosomal RNA are similar for 30S subunits reconsti tuted with either recombinant proteins or proteins isolated as a mixture fr om ribosomal subunits as well as for natural 30S subunits.