Charles L. Harris

tRNA sulfurtransferase: A member of the aminoacyl-tRNA synthetase complex in rat liver

Abstract Transfer RNA sulfurtransferase, tRNA methyltransferase, and aminoacyl-tRNA synthetase activity are associated in a complex in rat liver, which is excluded from Sephadex G-200 columns. The complex can also be isolated by subjecting cell supernatants to further centrifugation at 160,000 x g for 18 hours. The resulting pellet contains 70% of the total sulfurtransferase activity, and a 3-fold increase in specific activity is accomplished through pelleting. The data suggest that the enzymes of tRNA metabolism are organized in a large complex in rat liver.

A rapid diethylaminoethyl paper disk assay for transfer RNA sulfurtransferase.

A rapid assay for tRNA sulfurtransferase from Escherichia coli was developed, reducing the time needed to determine enzyme activity from 11 to 2 h. The reaction measured is the transfer of sulfur from [35S]cysteine to acceptor sites in a thionucleotide-deficient tRNA substrate. Processing is done by binding the product, [35S]-tRNA, to DEAE-cellulose filter disks. The disks are then treated to remove unreacted [35S]cysteine, cysteine-protein adducts and [35S]cysteinyl-tRNA. The DE81 disk assay and the 11-h standard assay are shown to give identical values over a wide range of incubation times and enzyme levels. Incorporation was greater when thionucleotide-deficient tRNA was used as substrate, as compared to fully modified tRNA. [35S]-tRNA was found to be the major reaction product, although some [35S]cysteine was also bound to the filters. The major thionucleoside labeled in nucleoside digests was 4-thiouridine, as determined by Bio-Gel P2 chromatography. We also observed other labeled peaks by this method, in amounts too small for positive identification. This rapid assay should be useful in the purification and study of this uncharacterized class of tRNA modification enzymes.

Cysteine and growth inhibition of Escherichiacoli: Derepression of the ilvGEDA operon

Abstract Cysteine inhibits growth of Escherichia coli by blocking isoleucine synthesis, presumably at the level of threonine deaminase (Harris, C.L. 1981. J. Bacteriol. 145 , 1031–1035). We show here that treatment of growing cultures with cysteine causes a decrease in the % charging of tRNAile, in proportion to the extent of growth inhibition. This effect is accompanied by a partial derepression of the ilvGEDA operon. These findings suggest a new way to limit isoleucine synthesis in E. coli which may be useful for studies of regulation of the ilvGEDA operon.

Isoleucyl-tRNA synthetase inactivation and the extent of aminoacylation of tRNAIle from Escherichia coli.

A difference in isoleucine acceptance between normal and sulfur-deficient tRNA from Escherichia coli C6 (rel-, met-, cys-) was eliminated when more isoleucyl-tRNA synthetase was added at the reaction plateau. Enzymatic deacylation was similar for both tRNAs. These results suggest that enzyme inactivation caused a premature reaction plateau which was not predicted by the rates of acylation and deacylation.

Modified nucleosides and the chromatographic and aminoacylation behavior of tRNAIle from Escherichia coli C6

Transfer RNA from Escherichia coli C6, a Met-, Cys-, relA- mutant, was previously shown to contain an altered tRNA(Ile) which accumulates during cysteine starvation (Harris, C.L., Lui, L., Sakallah, S. and DeVore, R. (1983) J. Biol. Chem. 258, 7676-7683). We now report the purification of this altered tRNA(Ile) and a comparison of its aminoacylation and chromatographic behavior and modified nucleoside content to that of tRNA(Ile) purified from cells of the same strain grown in the presence of cysteine. Sulfur-deficient tRNA(Ile) (from cysteine-starved cells) was found to have a 5-fold increased Vmax in aminoacylation compared to the normal isoacceptor. However, rates or extents of transfer of isoleucine from the [isoleucyl approximately AMP.Ile-tRNA synthetase] complex were identical with these two tRNAs. Nitrocellulose binding studies suggested that the sulfur-deficient tRNA(Ile) bound more efficiently to its synthetase compared to normal tRNA(Ile). Modified nucleoside analysis showed that these tRNAs contained identical amounts of all modified bases except for dihydrouridine and 4-thiouridine. Normal tRNA(Ile) contains 1 mol 4-thiouridine and dihydrouridine per mol tRNA, while cysteine-starved tRNA(Ile) contains 2 mol dihydrouridine per mol tRNA and is devoid of 4-thiouridine. Several lines of evidence are presented which show that 4-thiouridine can be removed or lost from normal tRNA(Ile) without a change in aminoacylation properties. Further, tRNA isolated from E. coli C6 grown with glutathione instead of cysteine has a normal content of 4-thiouridine, but its tRNA(Ile) has an increased rate of aminoacylation. We conclude that the low content of dihydrouridine in tRNA(Ile) from E. coli cells grown in cysteine-containing medium is most likely responsible for the slow aminoacylation kinetics observed with this tRNA. The possibility that specific dihydrouridine residues in this tRNA might be necessary in establishing the correct conformation of tRNA(Ile) for aminoacylation is discussed.