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H G Wittmann

Publications and source records attributed to H G Wittmann.

At least 19 recordsLinked to original sources
PubMed1991

Synthesis of a radioactive labeled undecagoldcluster for application in X-ray structure analysis of ribosomes.

In order to quantitate and to determine the extent of binding of a heavy atom derivative to ribosomal particles and other components of protein biosynthesis, we prepared a 14C-radioactive labeled monofunctional undecagoldcluster. This cluster can be bound either to exposed sulfhydryl groups at the surface of the ribosomes and its subunits, or to high nucleophilic amino groups of nucleic acids as well as to proteins.

Carbon Radioisotopes↗
PubMed1990

Characterization and preliminary crystallographic studies on large ribosomal subunits from Thermus thermophilus.

Diffracting crystals, suitable for X-ray crystallographic analysis, have been obtained from large (50 S) ribosomal subunits from Thermus thermophilus. These crystals, with P4(1)2(1)2 symmetry and a unit cell of 495 A x 495 A x 196 A, reach typically a size of 0.15 mm x 0.25 mm x 0.35 mm. Using synchrotron radiation at cryo-temperature, these crystals diffract X-rays to better than 9 A resolution, and do not show any measurable decay after a few days of irradiation. They complete a series of crystals, grown by us, from ribosomal particles of the same source, including a 30 S subunits, 70 S ribosomes and complexes of the latter with: (1) an oligomer of 35 uridine residues and (2) the same oligonucleotide together with approximately two Phe-tRNA(Phe) molecules. Crystallographic analysis of the various members of this series should provide information for investigating the conformational changes that take place upon the association of ribosomes from their subunits as well as upon binding of non-ribosomal components that participate in protein biosynthesis.

Crystallization↗
PubMed1990

Low-resolution models for ribosomal particles reconstructed from electron micrographs of tilted two-dimensional sheets.

Models of the whole ribosome (70S) and its large subunit (50S) were obtained at low resolution (47 and 28 A respectively) by three-dimensional image reconstruction using diffraction data collected from electron micrographs of two-dimensional ordered arrays. The comparison of the various reconstructed images, using interactive computer graphics, enabled the assessment of the reliability of the method, the derivation of the shape of the small subunit (30S) and the assignment of several functional features such as the probable path taken by the nascent protein chain, the presumed site for the process of biosynthesis of proteins, and a feasible mode for tRNA binding. The reconstructed models of the various ribosomal particles may be used for phasing of X-ray diffraction data at low resolution.

Chemical Phenomena↗
PubMed1990

Crystals of complexes mimicking protein biosynthesis are suitable for crystallographic studies.

A complex of 70S ribosomes from Thermus thermophilus together with an average of 1.5-1.8 equivalents of PhetRNA(Phe) and a short mRNA chain, composed of 35 +/- 5 uridines, was crystallized under the conditions used for the growth of crystals of isolated ribosomes from the same source. Considering the reproducibility of their growth, their internal order and their shape, the crystals of the complex are superior to those of isolated ribosomes. In accord with previous three-dimensional reconstruction and modeling experiments, we conclude that the complex is less flexible and that an average population of complexes is more homogeneous than that of isolated 70S ribosomes. The crystals of the complex diffract to higher than 15 A resolution and can be irradiated with synchrotron X-ray beam at cryo-temperatures for days without noticeable decay. Since the crystals of the complex are apparently isomorphous with these of the isolated 70S ribosomes (P4(1)2(1)2; a = b = 526; c = 315 A), they should provide tool for phasing as well as for locating the mRNA and tRNA binding sites.

Computer Graphics↗
PubMed1978

Ribosome structure.

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Amino Acid Sequence↗
PubMed1977

Improved electrophoretic and immunochemical techniques for the identification and characterization of mutant proteins, applied to ribosomal protein S8 in Escherichia coli mutants.

The ribosomal proteins of 11 mutants which are sensitive to starvation at elevated temperature and of 36 transductants derived from them were studied with several electrophoretic, immunochemical and proteinchemical methods. The following results were obtained: (1) Ribosomal protein S8 is altered in three of these mutants. (2) The amino acid exchange in proteins S8 of mutant N4128 is Glu leads to Lys in position 59 of the protein chain. (3) Temperature sensitivity and inability to recover from starvation at elevated temperatures are caused by the same mutational event which is, however, unrelated to the alteration in protein S8. Several electrophoretic and immunological procedures were applied during the characterization of these mutants. A modified immunoelectrophoresis on cellulose acetate gels was developed, and proved to be the most applicable procedure for the detection of mutationally altered ribosomal proteins. This procedure may gain general importance for detecting mutational alterations in other proteins.

Amino Acid Sequence↗
PubMed1977

Cold-sensitive growth of a mutant of Escherichia coli with an altered ribosomal protein S8: analysis of revertants.

26 cold-resistant revertants of a cold-sensitive Escherichia coli mutant with an altered ribosomal protein S8 were analyzed for their ribosomal protein pattern by two-dimensional polyacrylamide gel electrophoresis. It was found that 16 of them had acquired the apparent wild-type form of protein S8, one exhibits a more strongly altered SC than the original mutant and two revertants regained the wildtype form of S8 and, in addition, possess alterations in protein L30. The ribosomes of the residual revertants showed no detectable difference from those of the parental S8 mutant. The mutation leading to the more strongly altered S8 was genetically not separable from the primary S8 mutation; this indicates that both mutations are very close to each other or at the same site. The structural gene for ribosomal protein L30 was mapped relative to two other ribosomal protein genes (for proteins S5 and S8) by the aid of one of the L30 mutants: The relative order obtained is: aroE....rpmD(L30)....rpsE(S5)....rpsH(S8)....rpsL(S12). The L30 mutation impairs growth and ribosomal assembly at 20 degrees C and is therefore the first example of a mutant with defined 50S alteration that has (partial) cold-sensitive ribosome assembly. A double mutant was constructed which possesses both the S8 and the L30 mutations. It was found that the L30 mutation had a slight antagonistic effect on the growth inhibition caused by the S8 mutation. Thus the L30 mutants might have possibly arisen from the original S8 mutant first as S8/L30 double mutants which was followed by the loss of the original S8 lesion.

Cold Temperature↗
PubMed1977

Erythromycin resistant mutations in Bacillus subtilis cause temperature sensitive sporulation.

All of several hundred erythromycin resistant single site mutants of Bacillus subtilis W168 are temperature senstive for sporulation. The mutants and wild type cells grow vegetatively at essentially the same rates at both permissive (30 degrees C) and nonpermissive (47 degrees C) temperatures. In addition cellular protein synthesis, cell mass increases and cell viabilities are similar in mutant and wild type strains for several hours after the end of vegetative growth (47 degrees C). in the mutants examined, the temperature sensitive periods begin when the sporulation process is approximately 40% completed, and end when the process is 90% completed. At nonpermissive temperatures, the mutants produce serine and metal proteases at 50% of the wild type rate, accumulate serine esterase at 16% of the wild type rate, and do not demonstrate a sporulation related increase in alkaline phosphatase activity. The eryR and spots phenotypes cotransform 100%, and cotransduce 100% using phage PBS1. Revertants selected for ability to sporulate normally at 47 degrees C (spot), simultaneously regain parental sensitivity to erthromycin. No second site revertants are found. Ribosomes from eryR spots strains bind erythromycin at less than 1% of the wild type rate. A single 50S protein (L17) from mutant ribosomes shows an altered electrophoretic mobility. Ribosomes from spo+ revertants bind erythromycin like parental ribosomes and their proteins are electrophoretically identical to wild type. These data indicate that the L17 protein of the 50S ribosomal subunit from Bacillus subtilis may participate specifically in the sporulation process.

Bacillus subtilis↗