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Location of aromatic amino acids and belix content in Escherichia coli ribonucleic acid polymerase.

ABSTRACT: 1. The perturbing effect of glycerol on the direct spectrum of Escherichia coli DNA-dependent RNA polymerase has been studied. 2. By comparison with model compounds and with the unfolded polymerase in 3.8m-urea it was possible to determine the ratio of tyrosine and tryptophan residues present. On reduction of the urea-treated enzyme with 2-mercaptoethanol, no further change in the difference spectrum occurred. 3. The amino acid composition of the enzyme is given. 4. In the intact protein approx. 30% of the tryptophan and 54% of the tyrosine residues were exposed. In conjunction with the extinction value and molecular weight this corresponded to 7 tryptophan residues and 57 tyrosine residues on the surface and 16 tryptophan residues and 48 tyrosine residues ;buried'. 5. The optical rotatory dispersion of the enzyme was unaffected by 20% glycerol. 6. The helix content calculated from Moffit plots over 560-300nm was 13%, and from the 233nm trough 13%.

SUBMITTER: Nicholson BH

PROVIDER: S-EPMC1176905 | biostudies-other | 1971 Jun

REPOSITORIES: biostudies-other

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Project description:1. The technique of DNA-RNA hybridization was used to follow changes in the amount and average lifetime of unstable messenger RNA in Escherichia coli M.R.E. 600 over a wide range of different growth conditions. The method of analysis was based on the kinetics of incorporation of exogenous labelled nucleic acid bases into the RNA of steadily growing cultures, as described by Bolton & McCarthy (1962). 2. The ratio of the average lifetime of messenger RNA to the mean generation time of E. coli cultures was constant over the temperature range 25-45 degrees C in a given medium, but the constant varied with the nature of the growth medium. For cultures growing in sodium lactate-salts or glucose-salts media the ratio was 0.046+/-0.005 and in enriched broth it was 0.087+/-0.009. Measurements of the amounts of transfer RNA, ribosomal RNA and messenger RNA were also made. The results confirmed earlier reports that the ratio of the amount of messenger RNA to the amount of ribosomes in the cells is virtually constant. On the other hand, the ratio of the amount of transfer RNA to the amount of ribosomal RNA decreased with increasing growth rate at a given temperature. 3. In cultures at temperatures higher than necessary for optimum rates of growth the average lifetime of messenger RNA lengthened in harmony with the increased time required for cell division. It seems that suboptimum growth rates at higher temperatures cannot be explained simply as a combination of increased rates of synthesis and breakdown of messenger RNA with a grossly decreased efficiency of translation. The absolute rate of messenger RNA synthesis was lowered, and its amount in the cells was typical of all other cultures grown at lower temperatures in the same medium. 4. The rate of entry of exogenous labelled uracil into unstable messenger RNA and stable ribosomal RNA was constant in all media at all temperatures in the approximate ratio 1:2. In media supporting a lower rate of growth, e.g. lactate-salts or glucose-salts media, the messenger RNA fraction constituted 2.2+/-0.3% of the total cellular RNA. In enriched broth 3.6+/-0.3% of the total RNA was messenger.

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Location of aromatic amino acids and belix content in Escherichia coli ribonucleic acid polymerase.

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