Gisela Nass

Molecular weight distribution of the aminoacyl-tRNA-synthetases of Escherichia coli by gel filtration

SummaryMolecular weights of 18 aminoacyl-tRNA-synthetases of E. coli were estimated by gel filtration on Sephadex G-200.Molecular weights calculated from S20 and D20 values are known for four of the aminoacyl-tRNA-synthetases, the phenylalanyl- (Stulberg, 1967), isoleucyl- (Baldwin and Berg, 1966), tyrosyl- (Calendar and Berg, 1966) and the arginyl-tRNA-synthetase (Stöffler and Nass, 1967). These enzymes have been used as marker proteins for determination of the molecular weights of further 14 aminoacyl-tRNA-synthetases:1.Nine aminoacyl-tRNA-synthetases have a molecular weight between 90,000 and 120,000, i.e. the asparagyl-, asparaginyl-, glutaminyl-, glycyl-, histidyl-, leucyl-, lysyl-, seryl-and valyl-tRNA-synthetase.2.Alanyl-tRNA-synthetase: mol. wt. of 180,000.3.Methionyl- and threonyl-tRNA-synthetase: mol. wt. of 145,000.4.Prolyl- and glutamyl-tRNA-synthetase: mol. wt. of 75,000. The reliability of the used gel filtration for molecular weight determination is discussed.

Genetics of borrelidin resistant mutants of Saccharomyces cerivisiae and properties of their threonyl-tRNA-synthetase.

SummaryTwenty-two borrelidin resistant mutants of Saccharomyces cerivisiae were isolated, studied genetically and their threonyl-tRNA-synthetase was investigated. The borrelidin resistant mutants are classified into four groups. In the first group borrelidin resistance is coupled to the gene HOM3 coding for aspartokinase, in the second group to the gene LEU1. The borrelidin resistance in group three and four is not coupled to anyone of the genetic markers tested. Borrelidin resistance exhibited dominant behavior in all mutants except in the mutant of group 4. The properties of the ThrRS of the mutants of group one, two and four was found to be like the ones of the wild types. However the mutants of group three exhibit a structurally altered ThrRS, which is no longer inhibited by borrelidin.

Regulation of histidine biosynthetic enzymes in a mutant of Escherichia coli with an altered histidyl-tRNA synthetase.

SummaryA mutant of Escherichia coli was isolated that grew at a normal rate in minimal medium at 26°C, grew at a normal rate in minimal medium at 37°C only if exogenous histidine was supplied, and grew more slowly than normal at 42°C even in the presence of histidine. In very rich media the growth rate of the mutant was normal at 26°C and 30°C, but not at 37°C or 42°C. It may be described as a temperature-conditional histidine bradytroph with a decreased ceiling to its growth rate.The histidyl-tRNA synthetase of the mutant was found to be abnormal; in crude extracts the enzyme activity was less stable and had approximately a tenfold higher apparent KMfor histidine than normal.Under many growth conditions the histidine biosynthetic enzymes in the mutant were derepressed several hundred fold compared to the wild strain, even in the presence of exogenous genous histidine. In general, the degree of derepression in the mutant was proportional to the difference in growth rate between the mutant and normal strains; this relationship, however, did not hold below 30°C or above 37°C.The properties of the mutant could be related to the properties of its histidyl-tRNA synthetase by assuming that the enzyme participates both in protein synthesis and in histidine biosynthetic enzyme regulation and that at low temperature it functions relatively more effectively in protein synthesis than in repression, while at high temperature it functions relatively more effectively in repression.

Effect of borrelidin on the threonyl-tRNA-synthetase activity and the regulation of threonine-biosynthetic enzymes in Saccharomyces cerivisiae

SummaryIn yeast cells grown in the presence of Borrelidin the threonyl-tRNA-synthetase is inhibited and a specific derepression of the aspartokinase takes place. These results are compatible with the assumption that in yeast cells the threonyl-tRNA-synthetase is involved in the repression of the formation of the threonine biosynthetic enzyme.

Alteration of structure or level of threonyl-tRNA-synthetase in borrelidin resistant mutants of E. Coli

Borrelidin inhibits the growth of E. coli effectively [ 1, 21. Since no other biochemical reaction is known to be attacked directly by Borrelidin than the enzymatic activity of the threonyl-tRNA-synthetase (ThRS) [2, 31, we isolated Borrelidin-resistant mutants of E. coli K12 and E. coli B with the aim of finding mutants with an altered structure or level of ThRS. The ThRS of fifteen Borrelidin-resistant mutants of each E. coli strain was investigated. The properties of the ThRS of two Borrelidin-resistant mutants of E. coli K12 has been analysed extensively [4] . Here now we describe the characterization of the ThRS of the remaining Borrelidin-resistant mutants. By means of determination of enzyme constants and antibody neutralisation curves it is shown that the Borrelidinresistant mutants can be divided into three groups: one group of mutants exhibits constitutively increased levels of wildtype ThRS, the second group structurally altered ThRS, and in a third group of mutants no alteration of the structure or level of ThRS could be detected. This suggests that Borrelidin-resistance in the latter mutants is due to some other reason than alteration of ThRS activity. Since it is known that the ThRS participates in the regulation of formation of the threonine biosynthetic enzymes [2, 51, the level of aspartokinase was also determined in the Borrelidin-resistant mutants grown in the presence and absence of Borrelidin.

Regulation of the formation of isoleucyl-tRNA-synthetase and the level of isoleucine biosynthetic enzymes in E. coli K12

SummaryDuring derepression of threonine deaminase and acetolactate synthetase due to valine deficiency—initiated by α-aminobutyric acid limited growth of E. coli K12 or by limited valine supply to an ilv/leu auxotroph of E. coli K12—no alteration of the specific activity of isoleucyl-tRNA-synthetase occurs. Leucine limited growth of the auxotroph, leading to an even higher derepression of the isoleucine biosynthetic enzymes, also does not affect the specific activity of isoleucyl-tRNA-synthetase. However, under growth conditions where the same degree of derepression of threonine deaminase is due to isoleucine deficiency, as in E. coli K12B or two valine resistant mutants thereof grown in the presence of valine, or in the auxotroph during growth-limiting isoleucine supply, a specific two- to three-fold derepression of the isoleucyl-tRNA-synthetase takes place. But there is no strict correlation between the degree of derepression of threonine deaminase due to isoleucine deficiency and the degree of derepression of isoleucyl-tRNA-synthetase, as especially shown in case of the valine resistant mutant Val R4 and Val R5 grown in the presence of valine.These results demonstrate that the rate of formation of isoleucyl-tRNA-synthetase and of threonine deaminase are not regulated by the same molecular devices and that a certain degree of isoleucine deficiency is a prerequisite for a derepression of isoleucyl-tRNA-synthetase.

Change of isoaccepting threonyl-tRNA constitutively increased level of threonyl-tRNA-synthetase in E. coli

The level of an aminoacyl-tRNA-synthetase in bacteria can be increased reactively in answer to specific environmental conditions or constitutively due to mutation (cf. [ 1,2] and [3--61). In neither case the molecular reactions determining the intracellular level of an aminoacyl-tRNA-synthetase are fully understood. We have described mutants of E. coli K12, in which the level of wildtype threonyl-tRNA-synthetase (ThrRS) is increased constitutively by a factor of three to ten, depending on the mutant [4,7]. Since in many respects aminoacyl-tRNA-synthetases and tRNA can be looked at as a functional unit, we investigated the tRNAThr in one of our mutants, which has a five-fold increased level of ThrRS. Here we show that this intracellular increase of wildtype ThrRS is accompanied by a specific change in the amount of the three threonine isoaccepting tRNA’s.

The Formation of Phenylalanyl-tRNA-synthetase Crossreacting Material in Temperature sensitive Mutants of E. coli

The physiological role of the aminoacyl-tRNA-synthetases has been presented by F. C. Neidhardt (1967). In his laboratory, as already mentioned, several temperature sensitive mutants of E. coli have been isolated which hardly grow at 37° but grow almost like the wildtype cells at 25° and which show no enzymatic activity of the phenylalanyl-tRNA-synthetase in vitro (cf. Neidhardt, 1966).