Mapping proteins of the 50S subunit from Escherichia coli ribosomes

Mapping of protein positions in the ribosomal subunits was first achieved for the 30S subunit by means of neutron scattering about 15 years ago. Since the 50S subunit is almost twice as large as the 30S subunit and consists of more proteins, it was difficult to apply classical contrast variation techniques for the localisation of the proteins. Polarisation dependent neutron scattering (spin-contrast variation) helped to overcome this restriction. Here a map of 14 proteins within the 50S subunit from Escherichia coli ribosomes is presented including the proteins L17 and L20 that are not present in archeal ribosomes. The results are compared with the recent crystallographic map of the 50S subunit from the archea Haloarcula marismortui.     

A large nucleoprotein fragment of the 50-S ribosomal subunit of Escherichia coli.

A large nucleoprotein fragment was isolated from a nuclease digest of Escherichia coli 50-S ribosomes and purified to gel electrophoretic homogeneity. Conditions were employed under which the fragmentation pattern was reproducible and the various fragment fractions were stable and maintained their sedimentation and electrophoretic properties throughout the several preparative and analytical procedures used. Fractions that appeared homogeneous in sucrose gradient centrifugation were found to be heterogeneous by gel electrophoresis. The large fragment was purified to homogeneity by preparative gel electrophoresis. It contained 21 proteins, the 5-S RNA, and two large oligonucleotides which together total about two thirds the molecular weight of the 23-S RNA. Because it can be prepared reproducibly in large quantities and because of its size and stability, the fragment appears suitable for functional and structural studies and as the starting material for further fractionation. An important contributing factor to the observed stability and reproducibility was the maintenance of an unchanging ionic environment. A single buffer was employed throughout all the procedures, and the fragments produced by nuclease digestion were dissociated from each other by heat rather than by changing the medium.     

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.     

Isolation of all proteins from the 50S subparticles of ribosomes of Escherichia coli

The paper proposes a method of preparative isolation of all proteins from the 50S subparticle of E. coli ribosomes. The method is based on (1) preliminary fractionation into protein groups and ribonucleoprotein particles by a consecutive treatment of the 50S particles with increasing LiCl concentrations, and (2) chromatographic separation of protein groups on DE- and CM-cellulose and gel-filtration of separate fractions. The method allows to obtain any protein required for studies in preparative amounts avoiding many chromatographic stages. A detailed scheme of isolation of all proteins is given together with quantitative data of yields of individual proteins calculated per 6 g of the 50S subparticles.     

Shape of protein L11 from the 50S ribosomal subunit of Escherichia coli.

Protein L11 from the 50S ribosomal subunit of Escherichia coli A19 was purified by a method using nondenaturing conditions. Its shape in solution was studied by hydrodynamic and low-angle x-ray scattering experiments. The results from both methods are in good agreement. In buffers similar to the ribosomal reconstitution buffer, the protein is monomeric at concentrations up to 3 mg/mL and has a molecular weight of 16 000-17 000. The protein molecule resembles a prolate ellipsoid with an axial ratio of 5-6:1 a radius of gyration of 34 A, and a maximal length of 150 A. From the low-angle x-ray diffraction data, a more refined model of the protein molecule has been constructed consisting of two ellipsoids joined by their long axes.     

Assembly map of the 50-S subunit from Escherichia coli ribosomes, covering the proteins present in the first reconstitution intermediate particle

Highly purified proteins and 23-S RNA from the 50-S subunit of Escherichia coli ribosomes were used to study the assembly dependences of the early assembly proteins. The proteins under observation and the RNA were incubated at 4 mM Mg2+ and 44 degrees C, the unbound proteins were separated by sucrose gradient centrifugation, the RNA . protein complex was precipitated with trichloroacetic acid, and the complex-bound proteins was identified by means of sodium dodecylsulfate gel electrophoresis. A systematic analysis led to the establishment of an assembly map including 17 proteins which represent the protein moiety of the first reconstitution intermediate particle.     

The synthesis of a photoreactive puromycin analogue and its application for labeling proteins in the 50-S subunit of Escherichia coli ribosomes.

A photoreactive puromycin analogue, 6-dimethylamino-9-[3-(p-azido-L-beta-phenylalanylamino)-3-deoxy-beta-ribofuranosyl] purine, was synthesized. Biological activity was demonstrated by inhibition of the poly (U)-directed phenylalanine-incorporation system and by decomposition of isolated polysomes from Escherichia coli. The 3H-labeled puromycin analogue was covalently attached to the 50-S subunit of isolated 70-S ribosomes from Escherichia coli after irradiation. More than 90% of the radioactivity was bound to the protein fraction. The 70-S proteins were separated by two-dimensional gel electrophoresis. The proteins labeled primarily were those of the 50-S subunit, identified as L6, L13, L18, L22 and L25. On the basis of the affinity label used and supportive data from the literature, it is concluded that these proteins are at the active center of the 50-S particle and probably belong to the region of the ribosomal A site.     

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.