H.G. Wittmann

Ribosomal proteins. VII. Two-dimensional polyacrylamide gel electrophoresis for fingerprinting of ribosomal proteins.

A two-dimensional polyacrylamide gel system has been developed, improving the analytical separation of complex protein mixtures as obtained from ribosomes. An equipment is described in detail (Figs. 1–4) which permits a simultaneous electrophoretic separation of five protein mixtures under identical conditions. By this method the protein mixtures of E. coli ribosomes can be resolved into about 50 components (Fig. 5). The use of this fingerprinting technique facilitates and accelerates considerably a number of investigations on the structure of ribosomes.

Ribosomal proteins: XXXIII. Location of amino-acid replacements in protein S12 isolated from Escherichia coli mutants resistant to streptomycin☆

Abstract Ribosomal protein S12, the protein coded by the strA ciatron, was isolated from nine streptomycin-resistant mutants originating from various Escherichia coli strains. Analysis of the tryptic peptides revealed that each mutant had a single amino-acid replacement in one of two peptides: in mutants belonging to the allele types strA 1, strA 2 and strA 60 the lysine residue in position 42 (peptide T6) of protein S12 is replaced by one of three ammo acids (asparagine, threonine or arginine) whereas the mutants belonging to allele type strA 40 have a replacement of lysine by arginine in peptide T15. There is a good agreement between our protein-chemical data and earlier genetic data on streptomycin-resistant mutants.

Characterization and crystallization of ribosomal particles from Halobacterium marismortui

Ribosomes and their subunits have been isolated from Halobacterium marismortui, an extremely halophilic bacterium from the Dead Sea. The stability and functional activity of the subunits were tested under a wide range of salt conditions. Three‐dimensional microcrystals of the large ribosomal subunits have been obtained. Electron microscopy of positively stained thin sections of these crystals showed that the particles are closely packed with approximate cell constants of 310 × 350 Å.

Cryocrystallography of ribosomal particles

Crystals suitable for X-ray study have been prepared from biochemically active ribosome particles or their complexes with tRNA and polypeptide chains. At ambient temperature the useful lifetime of these crystals under synchrotron irradiation is limited to a few minutes. However, upon cooling to cryogenic temperatures around 85 K, the original resolution limit (up to 4.5 A) can be recorded and radiation damage is virtually eliminated. Hence it has become possible to collect a complete data set from one single crystal. Crystals were cooled as rapidly as possible, either in a cold gas stream, or by immersion in liquid propane. Before cooling crystals were transferred either to an inert hydrocarbon environment, or to solutions similar to the crystallizing ones but with a higher viscosity. In several cases soaking in a cryosolvent was required. Crystallographic data were collected with intense synchrotron radiation. Full data sets have been measured for native and derivatized crystals of 50S ribosomal subunits from H. marismortui as well as from their complexes with tRNA and nascent polypeptide chains, from the wild type and a mutant of 50S subunits from B. stearothermophilus, and from crystals of native and derivatized 30S ribosomal subunits from T. thermophilus.

Ribosomal proteins. XXV. Immunological studies on Escherichia coli ribosomal proteins.

Abstract Antisera specific for each of the twenty-one 30 s proteins and for 24 50 s ribosomal proteins of Escherichia coli were prepared and used to investigate whether there are any extensive sequence homologies between the different proteins. No immunological cross-reactions were detected between any of the proteins, except L7 and L12 which gave complete cross-reaction. We conclude that the proteins investigated differ substantially in their sequences, but that proteins L7 and L12 have considerable structure homology.

International symposium on protein synthesis. Summary of Fogarty Center-NIH Workshop held in Bethesda, Maryland on 18-20 October, 1976.

+To whom reprint requests should be addressed The session devoted to initiation of protein synthesis in prokaryotes centered around two major problems. The most intriguing and so far the most difficult one is that of recognition by the ribosome of initiation signals in the messenger; the other is the assembly of initiation complexes, a process mediated by initiation factors and GTP. L. Bosch (Ieiden) reviewed the present status of the field in his opening comments. The postulate of Shine and Dalgarno (also see

Effect of different mutaions in ribosomal protein S5 of Escherichia coli on translational fidelity

SummaryThe effect of three different types of mutations in ribosomal protein S5 of Escherichia coli on translational fidelity has been studied. Two of them, namely that conferring resistance to spectinomycin and that selected for partial suppression of a temperaturesensitive alanyl-tRNA synthetase mutation, do not exhibit ribosomal ambiguity in the in vivo and in vitro test system employed. In constrast, mutations in ribosomal protein S5 selected for suppression of streptomycin dependence mutations are able to derestrict the restriction of translational ambiguity imposed by strA mutations, though to different degrees depending on the type of mutation. Mutants in which streptomycin dependence is suppressed by an alteration in protein S5 are more restrictive than mutants resistant to streptomycin. Again, the extent of restriction depends on the type of the strAd allele.In conclusion: mutations in ribosomal protein S5 can act as ram mutations like mutations in protein S4. The part of the S5 polypeptide involved in control of translational fidelity is different from regions altered in spectinomycin resistant strains and in the alanyl-tRNA synthetase suppressor mutant.

Active sites in Escherichia coli ribosomes

The model of active sites in E. coli ribosome illustrated in the figure is based on the presently available experimental results. It is far from being complete and should not be overinterpreted as an accurate topographical model. More data on the functional role of ribosomal components and on the topography of the subunits can be expected in the near future and will add to the knowledge on the active sites in ribosomes.

Single crystals of large ribosomal particles from Halobacterium marismortui diffract to 6 Å

Large, well-ordered three-dimensional crystals of 50 S ribosomal subunits from Halobacterium marismortui have been obtained by seeding. The crystals have been characterized with synchrotron X-ray radiation as monoclinic, space group P2(1), with unit cell dimensions of a = 182(+/- 5) A, b = 584(+/- 10) A, c = 186(+/- 5) A, beta = 109 degrees. At 4 degrees C, the crystals (0.6 mm X 0.6 mm X 0.1 mm) diffract to 6 A resolution and are stable in the synchrotron beam for several hours. Compact packing is reflected from the crystallographic unit cell parameters and from electron micrographs of positively stained thin sections of embedded crystals.

Crystallization of Escherichia coli ribosomes

The knowledge of the structure of the ribosome is an essential requirement to reveal its role at the molecular level in the process of protein biosynthesis. This information is being obtained by a battery of chemical, physical, immunological and genetic methods (for reviews see [1]). Important methods for the study of the three-dimensional structure of the ribosomes are X-ray crystallography and image reconstruction. Two-dimensional ordered sheets of ribosomes from a few eukaryotic species are found under special conditions in vivo [2-4] or in cell homogenates [5]. Small two-dimensional arrays of E. coli ribosomal subunits were obtained in vitro [6,7]. The first three-dimensional crystals of ribosomal particles were obtained in vitro from the large subunit of the Bacillus stearothermophilus ribosome [8,9]. These have been characterized by three-dimensional image reconstruction studies [10]. Here we report the in vitro crystallization of E. coli ribosomes. We have obtained ordered threedimensional microcrystals. After washing and redissolving, the crystalline ribosomes sediment with a coefficient of 70 S in a sucrose gradient and are biologically active in the poly(U) in vitro system. Electron micrographs of thin sections of the crystals show regular arrangement of the ribosomes, and their optical diffraction patterns extend out to about 6.5 nm. Thus three-dimensional image reconstruction studies can be carried out. 2. MATERIALS AND METHODS