Biogenesis of ribosomes: free ribosomal protein pools in Escherichia coli.

Abstract

Proteins from ribosomal subunits (30 s and 50 s) have been fractionated into split (SP-30 and SP-50) and core (core-30 and eore-50) proteins. Antisera prepared in rabbit against them are shown to be highly group-specific as judged by the Ouchterlony (1967) double diffusion test and by precipitin reaction in solution. Various parameters which influence the immuno-precipitation of these proteins by specific antisera have been investigated. It is demonstrated that under controlled conditions this provides a sensitive and reliable method for characterization and quantitative estimation of free ribosomal proteins. The technique has been successfully applied in the investigations of various properties of free ribosomal protein pools existing in Escherichia coli. It is concluded that free ribosomal proteins in E. coli may constitute 8 to 14% of total soluble proteins under different growth conditions. Relative pool sizes of four classes of proteins expressed as a percentage of the total soluble proteins in bacteria grown in L broth (doubling period, 30 min) are estimated to be core-30, 2.1; core-50, 3.4; SP-30, 3.6; SP-50, 5.0. From the studies on bacteria grown in different media (doubling period, 30, 42 and 60 min), we further conclude that the amount of free proteins increases with the growth rate so as to constitute a constant fraction (7 to 9%) of total ribosomal proteins. Relative pool-sizes corresponding to four classes of ribosomal proteins, however, remain unaltered by different growth rate. Studies on the time-course of incorporation of labelled amino acids show that the level of radioactivity (specific activity) in free ribosomal protein pools reaches a saturation value in 2 to 4 minutes with an average half-life of about 50 seconds. Kinetic curves (specific activity versus time) for free and bound ribosomal proteins are found to be compatible with a precursor-product relationship. Pulseand-chase experiments further suggested that there is no significant breakdown of proteins in pools and that they are quantitatively transferred to mature ribosomes. Relative pool-sizes and kinetic behaviour of free ribosomal proteins indicate that these proteins are synthesized in a co-ordinate manner. Various possible mechanisms for a co-ordinate synthesis of ribosomal RNA and proteins at the level of subunits and at the level of whole ribosomes are discussed.