Reconstitution of Escherichia coli 30 S Ribosomal Subunits from Purified Molecular Components

Abstract

Abstract Reconstitution of 30 S ribosomal subunits from 16 S RNA and a mixture of purified individual 30 S ribosomal proteins has been studied. Proteins from the 30 S ribosomal subunit of Escherichia coli were purified by a combination of phosphocellulose and DEAE-chromatography, and Sephadex gel filtration. The proteins purified correspond to the 21 proteins generally accepted as 30 S proteins, with the exception of two proteins, P3b and P3c, which correspond to the protein S6 studied by other workers. P3b and P3c are closely related, and one is probably a derivative of the other. Using a mixture of these purified proteins, reconstitution of functionally active 30 S subunits has been demonstrated. Reconstituted particles had higher activities in poly(U)-directed polyphenylalanine synthesis than reference 30 S particles in several experiments. The functional activity of reconstituted particles was also examined in several other assays; these included natural messenger RNA-directed polypeptide synthesis, poly(U)-directed Phe-tRNA binding, AUG-directed fMet-puromycin formation, AUG-directed fMet-tRNA binding, and the binding of termination codon UAA in the presence of chain termination factors. In all cases, activities comparable to reference 30 S subunits were observed. The sedimentation properties and the protein composition of reconstituted particles were also similar to 30 S ribosomal subunits. The kinetics of reconstitution using purified protein mixtures was essentially identical with those of reconstitution using unfractionated 30 S proteins. These results strongly suggest that 21 purified 30 S proteins together with 16 S RNA are sufficient to reconstitute 30 S subunits, and that no essential 30 S components were lost during the fractionation and purification of the 30 S proteins. Single component omission experiments indicated that all purified proteins, except P1(S1) and P3b,c(S6), are required for full functional activity. A requirement for P9a(S16) has been shown in some, but not all, experiments. P3b,c(S6) and P9a(S16) have been shown to be involved in the reconstitution reaction in other experiments (Mizushima, S., and Nomura, M. (1970) Nature 226, 1214; Nomura, M. (1973) Science 179, 864) and therefore are 30 S components. It is still not clear whether P1(S1) should be considered a true 30 S protein or a ribosomal-associated factor.