Robert M. Bock

Separation of labeled from unlabeled proteins by equilibrium density gradient sedimentation

Abstract If equilibrium density gradient ultracentrifuge experiments are conducted under carefully designed conditions, the resolution of the method can be markedly improved or, alternatively, the experiment can be designed to describe the nature and amount of many widely varying macromolecules in a single experiment. The theoretical limits on resolution are examined for two types of experiments. Solutes have been found which give high resolution and appropriate densities for protein, RNA, and DNA equilibrium centrifugation. The properties of these solutes are tabulated. The theory has been tested by separation of normal β-galactosidase from samples labeled with N 15 , C 13 , and deuterium. It is shown that the method will resolve labeled from unlabeled proteins if an adequate, but attainable, density difference has been introduced.

Specificity of sRNA for recognition of codons as studied by the ribosomal binding technique

Multiple sRNA species which are specific for individual amino acids have been purified from yeast and Escherichia coli sRNA using techniques of column chromatography or countercurrent distribution. The purified sRNA fractions, after charging with radioactive amino acid, have been tested for binding to ribosomes in the presence of the trinucleotides assigned as codons for the respective amino acids. From the stimulation of binding, it is concluded that (1) an sRNA species shows strict specificity for the recognition of the first letter of a codon; and (2) an sRNA species often can recognize multiple codons differing in the third letter. The total results, which are summarized in Table 4, appear to provide support for the postulates of the wobble hypothesis ( Crick, 1966 ).

Studies on polynucleotides: LXXV. Specificity of tRNA for codon recognition as studied by the ribosomal binding technique☆☆☆

Abstract Multiple species of transfer RNA specific for individual amino acids from Escherichia coli and yeast were tested for their binding to ribosomes in the presence of the trinucleotides known to be codons for the respective amino acids. From the stimulation of binding, it is concluded that tRNA species can often recognize multiple codons differing in the third letter. The total results (including our previous work) on the recognition patterns of E. coli and yeast transfer RNAs which are summarized in Table 4, are in agreement with the postulates of the Wobble hypothesis (Crick, 1966) and show interesting differences in the recognition of a synonym codon set by transfer RNAs from yeast and E. coli .

Cytokinin Activity: Localization in Transfer RNA Preparations

Transfer RNA from yeast, liver, and Escherichia coli has cytokinin activity in the tobacco callus bioassay, whereas ribosomal RNA from yeast is inactive. In contrast to fractions of yeast transfer RNA rich in serine acceptor and cytokinin activity, preparations (70 to 90 percent pure) of arginine transfer RNA2, glycine transfer RNA, phenylalanine transfer RNA, and valine transfer RNA1 and of highly purified alanine transfer RNA from yeast were inactive at concentrations of 20 to 2500 micrograms per liter. One molecule of 6-(γ,γ-dimethylallylamino) purine per 20 molecules of yeast tRNA would account for the observed cytokinin activity. The number of major molecular species contributing to cytokinin activity of transfer RNA, therefore, must be small.

The nucleases of yeast. II. Purification, properties and specificity of an endonuclease from yeast.

Abstract A nuclease has been purified approx. 30-fold from the supernatant fraction of a hybrid yeast (Saccharomyces fragilis × Saccharomyces dobzhanskii) by salt fractionation, chromatography on Sephadex G-200, and DEAE-cellulose chromatography. The optimum pH is 7.6 and Mg2+ is required for the full activity. The action of this nuclease on polyribonucleotides is exclusively endonucleolytic. The major products upon extensive digestion of homopolymers are di- and trinucleotides having 5′-phosphomonoester end groups. The formation of mononucleotide is slight. Polyadenylic acid and polyuridylic acid are hydbrolyzed first to a family of small oligonucleotides having 5′-phosphomonoester end groups. The distribution of these oligonucleotides is not completely random and depends on the conformation of the substrate. Hydrolysis of shorter chains is much slower than longer chains. This endonuclease has no apparent specificity for a particular base residue in polynucleotides. This mode of hydrolysis coupled with its high stability make this enzyme a very useful reagent for the preparation of oligonucleotides with 5′-phosphate ends from either synthetic polynucleotides or nature RNA. This enzyme appears to be specific for polynucleotides having a random coil conformation. Double- and triple-stranded helical conformations are less susceptible to attack. Polyadenylic acid, polyuridylic acid and polyinosinic acid are hydrolyzed much faster than polycytidylic acid, transfer RNA or ribosomal RNA. The enzyme preparation hydrolyzes denatured DNA at approximately the same rate as yeast ribosomal RNA. Highly polymerized native Escherichia coli DNA is almost inactive as substrate.

The spectrophotometric constants of di- and trinucleotides in pancreatic ribonuclease digests of ribonucleic acid

Abstract The di- and trinucleotides arising from the digestion of RNA with pancreatic ribonuclease were isolated by a combination of column and paper chromatography. The spectra of the oligonucleotides at pH 1, 7, and 11 were determined and from these data the spectrophotometric constants at 20° were calculated. These values were found to be a function of temperature and representative dependencies are given.

Covalent integrity and molecular weights of yeast ribosomal ribonucleic acid components

Abstract 1. Yeast ribosomal RNA has been isolated from 83-S particles with sodium dodecyl sulfate, Macaloid (an anionic clay), and phenol. 2. RNA solutions were heated to 80° for 1 min in order to dissociate polynucleotide fragments held together by non-covalent forces. More than 60 % of the 26-S RNA in the several preparations tested survived this treatment and is thus judged to consist of intact molecules. 3. The molecular weights of the 17-S and 26-S RNA species were estimated from sedimentation equilibrium data. The two components were found to contain approximately 1800 and 3300 nucleotides, respectively.

Cytokinins: Isolation and identification of 6-(3-methyl-2-butenylamino)-9-β-D-ribofuranosylpurine (2iPA) from yeast cysteine tRNA

Abstract A cytokinin in yeast cysteine tRNA has been isolated as the riboside and has been shown to have uv and mass spectra identical with those of synthetic 6-(3- methyl-2-butenylamino)-9-β - D -ribofuranosylpurine.

Physicochemical studies of fatty acid synthetase a multienzyme complex from pigeon liver

Summary The molecular weight of pigeon-liver fatty acid synthetase was calculated from the s/D ratio and from separately determined diffusion and sedimentation coefficients. Values of 5.67·10 5 and 5.33·10 5 , respectively, were obtained. This multi-enzyme complex is converted to components with lower sedimentation coefficients in buffer of low ionic strength and in solutions of high pH.

An analysis of arginine codon multiplicity in rabbit hemoglobin

Two arginyl tRNAs from yeast (Arg tRNA I and Arg tRNA II) are separable by chromatography. Arg tRNA I which binds to the Escherichia coli ribosomes in response to the triplets CpGpU, CpGpC and CpGpA, transfers arginine only into the C-terminal position of the α -chain of rabbit hemoglobin (tryptic peptide α T17, position 141). Arg tRNA II, which binds to E. coli ribosomes in response to the triplets ApGpA and ApGpG, transfers arginine into position 31 of the α -chain (corresponding to peptide α T4). Codon assignments to positions 31 and 141 of the α -chain, based on the utilization of arginine from either of the two tRNA fractions, are consistent with known amino acid replacements at these positions, and could have been produced by a change of a single base. No labeled arginine was transferred into position 92 (corresponding to peptide α T11) nor into the two β -chain arginine positions. The lack of labeling of position 92 may be due to its specification by the triplet CpGpG, since neither of the tRNA fractions was bound to ribosomes in the presence of CpGpG. This codon assignment is consistent with the observed replacement of arginine at position 92 by leucine and glutamine in mutant human hemoglobins. The lower level of β -chains synthesis precluded a determination of the transfer specificity of β -chain arginine residues. Nevertheless, only the triplet ApGpPu at positions 31 and 41 of the β -chain can be interrelated with observed amino acid replacements, by a change of a single base.