Altered bacteriophage T4 ribonucleic acid metabolism in a ribonuclease II-deficient mutant of Escherichia coli.

Some early T4 ribonucleic acids were not found in an infected ribonuclease II-deficient strain but were formed in ribonuclease II+ transductants as well as in wild-type Escherichia coli.     

Cleavage by ribonuclease III of the complex of 30 S pre-ribosomal RNA and ribosomal proteins of Escherichia coli

In lysates of Escherichia coli strain AB301-105, newly-formed 30 S pre-ribosomal RNA moved in a complex with protein, at 53 S. A 53 S particle also formed from the purified RNA and 30 S and 50 S ribosomal proteins, and could be cleaved by RNAase III to yield 30 S and 48 S particles.     

Mapping and characterization of a mutation in Escherichia coli that reduces the level of ribonuclease III specific for double-stranded ribonucleic acid.

Localization of a mutation affecting ribonuclease III activity (an enzyme specific for double-stranded ribonucleic acid) in Escherichia coli was attempted. By a series of matings and transduction experiments, the mutation rnc-105 was mapped near the nadB gene. In strains carrying this mutation, another mutation (ranA2074) was also found. Based on available data, their order on the E. coli chromosome appears to be tyrA, ranA, nadB, rnc, purI. Strains carrying either the ranA2074 or the rnc-105 mutation fail to grow at 45 C in enriched medium, whereas strains carrying only the rnc-105 mutation are defective in ribonuclease III activity. Strains carrying either of these mutations grow more slowly than corresponding wild-type strains in all media tested at all temperatures; the rnc-105 mutation reduces the growth rate more than the ranA2074 mutation. T4 and T7 bacteriophages form plaques with a lower efficiency on strains carrying the rnc-105 mutation than on other strains. Thus we suggest that ribonuclease III is beneficial for normal growth of E. Coli and that at higher temperatures it becomes indispensable.     

Degradation of ribonucleic acid by immobilized ribonuclease.

An immobilized enzyme (pancreatic ribonuclease bound to porous titania) was investigated for the degradation of purified yeast ribonucleic acid as a substrate. The immobilized enzyme is active and stable in the pH range 4--8. Dependence of enzymatic activity on ionic strength, pH, temperature, fluid flow rate, and substrate concentration were investigated. A cumulative fluid residence time of 6 sec is sufficient for 50% substrate conversion at 25 degrees C and pH 7.0. The critical flow rate (i.e., the fluid flow rate necessary to remove film diffusion resistance) approximately doubles with each 10 degree C rise in reaction temperature. The critical flow rates obtained in this study are about 40 times greater than those obtained for a similar study on immobilized glucose oxidase. Arrhenius plots gave activation energies of -9.6 and -7.1 kcal/g mol at pH 4.6 and 7.0, respectively. The work reported herein is a bench-scale investigation of an immobilized enzyme with primary emphasis on the mass transfer and kinetic characteristics of the system. The rapid reaction rates obtainable at relatively low temperatures offer a potential alternative method of purifying yeast single cell protein (SCP) with miminum loss of desired protein. The key questions are how such a system would react in a yeast homogenate, what conditions in such a system must be controlled, and what type of immobilized reactor should be utilized, if such further work continued to show promise.     

Escherichia coli mutant lacking 4-thiouridine in its transfer ribonucleic acid.

A mutant of Escherichia coli has been isolated that lacks 4-thiouridine, a rare base in transfer ribonucleic acid. The mutant grows at the same rate as wild-type cells. It shows little near-ultraviolet-induced growth delay, thus supporting earlier hypotheses that 4-thiouridine in transfer ribonucleic acid is the chromophore for this growth delay.     

The aminoacylation of transfer ribonucleic acid. Recognition of methionine by Escherichia coli methionyl-transfer ribonucleic acid synthetase.

The mechanism of the recognition of methionine by Escherichia coli methionyl-tRNA synthetase was examined by a kinetic study of the recognition of methionine analogues in the ATP-PPi exchange reaction and the tRNA-aminoacylation reaction. The results show that the recognition mechanism consists of three parts: (1) the recognition of the size, shape and chemical nature of the amino acid side chain at the methionine-binding stage of the reaction; (2) the recognition of the length of the side chain at the stage of aminoacyl-adenylate complex-formation; (3) the recognition of the sulphur atom in the side chain at the stage of methionyl-tRNA formation. It is proposed that the sulphur atom interacts with the enzyme to induce a conformational change. A model of the active site incorporating the mechanism of methionine recognition is presented.