Modified nucleosides and the chromatographic and aminoacylation behavior of tRNA(Ile) 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.
Harris
C L
CL
Department of Biochemistry, West Virginia University, School of Medicine, Morgantown 26506.
Marashi
F
F
Sakallah
S
S
D002845:Chromatography; D003545:Cysteine / Q000502:physiology; D004926:Escherichia coli / Q000502:physiology*; D005978:Glutathione / Q000502:physiology; D007533:Isoleucine-tRNA Ligase / Q000378:metabolism; D012329:RNA, Bacterial; D012345:RNA, Transfer, Amino Acid-Specific / Q000502:physiology; D012346:RNA, Transfer, Amino Acyl; D012355:RNA, Transfer, Ile / Q000502:physiology; D012263:Ribonucleosides / Q000032:analysis; D013455:Sulfur / Q000378:metabolism; D013873:Thionucleotides / Q000378:metabolism; D046249:Transfer RNA Aminoacylation
D012329:RNA, Bacterial; D012345:RNA, Transfer, Amino Acid-Specific; D012346:RNA, Transfer, Amino Acyl; D012355:RNA, Transfer, Ile; D012263:Ribonucleosides; D013873:Thionucleotides; D013455:Sulfur; D007533:Isoleucine-tRNA Ligase; D005978:Glutathione; D003545:Cysteine