Exchange of ribosomal proteins among the ribosomes of Escherichia coli.

Summary The exchange of ribosomal proteins among ribosomes of E. coli has been measured, using a density label technique. As expected most of the proteins do not exhange appreciably. However a substantial fraction of each of proteins S1, S2, S21, L7/L12, L9, L10, L11, L26 and L33 is found to exchange, but exchange of S1, S2, L7/L12, L10, L11 and L26 is found to occur in vitro after lysis of the cells, and therefore it is not possible to say whether or not these proteins also exchange in vivo. In contrast S21, L9 and L33 do not exchange after lysis of the cells and we therefore conclude that these proteins exchange in vivo. The maximum level of exchange of S21, L9 and L33 is attained so rapidly that we were unable to show whether or not it was dependent on protein synthesis.     

Selective spin-labeling of the ribosomal proteins of 70S ribosomes from Escherichia coli.

We have used a series of N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl) maleimide spin labels of different length to label, covalently and selectively, the most reactive sulfhydryl groups of 70S ribosomal proteins of Escherichia coli. Under short periods of labeling (1--2 min), less than two spin labels per ribosome are incorporated and were shown to be distributed mainly on five ribosomal proteins in the following order: S18 greater than S21, L27 greater than S17, and S12. With a long period of labeling (3 h) up to 13 spin labels are attached to the ribosome, and protein S1 is the most labeled. The shape of the electron paramagnetic resonance (epr) signal shows two components with a predominance for the strongly immobilized orientation, and the percentage of these components in each spectra has been evaluated. When the distance between the nitroxide group and the maleimide-attaching group exceeds 6 A (1 A = 0.1 nm) the strongly immobilized orientation disappears. The effect of magnesium ions on these selectively spinlabeled ribosomes shows that the dissociation into subunits does not affect the epr signal, but more spin labels are incorporated into the subunits if labeling is performed under conditions of dissociation.     

Analysis of kethoxal bound to ribosomal proteins from Escherichia coli 70S reacted ribosomes

To investigate ribosome topography and possible function, 70S ribosomes of $\text{Escherichia coli}$ were reacted with the dicarbonyl compound kethoxal. Ribosomal protein was extracted after reaction, and through two dimensional gel electrophoresis, the reactive proteins of the two subunits were identified. From the 30S subunit, the most reacted proteins were S2, S3, S4, S5 and S7 and from the 50S subunit, L1, L5, L16, L17, L18 and L27. The results with kethoxal are compared with other modifiers of ribosomal proteins.     

On the statistical significance of homologous structures among the Escherichia coli ribosomal proteins

Summary Completion of the sequence determination of all 52 Escherichia coli ribosomal proteins enabled a final comparison of their sequences. Similarities in amino acid compositions were compared to the relatedness of the sequences, which was analyzed statistically with the aid of the computer programs RELATE and ALIGN.Among the examined 52×52 possible protein pairs at least 40 pairs were found that can be regarded as distantly related (showing segment comparison score values slightly above 3.0 S.D. units). These protein pairs were further examined with the programs ALIGN and SEEK to locate homologous sequence stretches. In no case were two complete homologous sequences found (with the exception of the known identical pairs L7/L12 and S20/L26). However, short homologous sequence regions were observed. Beside those protein pairs that show significant although distant relatedness, other pairs were slightly below the threshold value of 3.0 S.D. units.Those pairs observed to be distantly related consisted either of two proteins from the same subunit or of one protein from each of the different subunits. A further analysis of these pairs revealed a correlation between their relatedness and their time of incorporation into the ribosome during assembly.     

500-MHz 1H-NMR studies of ribosomal proteins isolated from 70-S ribosomes of Escherichia coli

A method for the large-scale isolation of ribosomal proteins is described avoiding pre-separation of 30-S and 50-S subunits. Five proteins isolated in this way were studied with high-resolution 1H NMR at 500 MHz. These are S21, L18, L25, L30 and L33. The results show that L18, L25 and L30 exhibit tertiary structure in solution and indications for secondary structure in S21 are found. Protein L33 appears to be a random coil. Several resonances in the 1H NMR spectra are assigned to particular protons of amino acid residues, e.g. the aromatic ring protons of tyrosines and histidines, and epsilon-protons of lysines.     

Biogenesis of ribosomes: free ribosomal protein pools in Escherichia coli

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 non-specific binding of ribosomal RNA to Escherichia coli ribosomes

Ribosomal RNA (rRNA) binds to ribosomes which contain small pieces of endogenous messenger RNA, when assayed by sticking to Millipore filters. This association is lost when the messenger RNA is removed by incubation of the ribosomes in buffer containing 10⁻⁴ m-Mg²⁺, but can be recovered by the addition of turnip yellow mosaic virus (TYMV) RNA. TYMV RNA promotes the binding of equal masses of 16 and 23 s rRNA to ribosomes. The incubation in 10⁻⁴ m-Mg²⁺ buffer, which destroys the ability of ribosomes to bind rRNA, increases their ability to bind TYMV RNA fivefold. Free rRNA does not compete with ribosomes for ribosome-binding sites in TYMV RNA. These results suggest that the binding of messenger RNA to free rRNA does not reflect the binding of messenger RNA to ribosomes.