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.     

Studies on chemical modification of thionucleosides in the transfer ribonucleic acid of Escherichia coli

(35)S-labelled tRNA from Escherichia coli was treated with chemical reagents such as CNBr, H(2)O(2), NH(2)OH, I(2), HNO(2), KMnO(4) and NaIO(4), under mild conditions where the four major bases were not affected. Gel filtration of the treated tRNA showed desulphurization to various extents, depending on the nature of the reagent. The treated samples after conversion into nucleosides were chromatographed on a phosphocellulose column. NH(2)OH, I(2) and NaIO(4) reacted with all the four thionucleosides of E. coli tRNA, 4-thiouridine (s(4)U), 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U), 2-thiocytidine (s(2)C) and 2-methylthio-N(6)-isopentenyladenosine (ms(2)i(6)A), to various extents. CNBr, HNO(2) and NaHSO(3) reacted with s(4)U, mnm(5)s(2)U and s(2)C, but not with ms(2)i(6)A. KMnO(4) and H(2)O(2) were also found to react extensively with thionucleosides in tRNA. Iodine oxidation of (35)S-labelled tRNA showed that only 6% of the sulphur was involved in disulphide formation. Desulphurization of E. coli tRNA with CNBr resulted in marked loss of acceptor activities for glutamic acid, glutamine and lysine. Acceptor activities for alanine, arginine, glycine, isoleucine, methionine, phenylalanine, serine, tyrosine and valine were also affected, but to a lesser extent. Five other amino acids tested were almost unaffected. These results indicate the fate of thionucleosides in tRNA when subjected to various chemical reactions and the involvement of sulphur in aminoacyl-tRNA synthetase recognition of some tRNA species of E. coli.     

Three photo-cross-linked complexes of yeast phenylalanine specific transfer ribonucleic acid with aminoacyl transfer ribonucleic acid synthetases.

Yeast tRNA-Phe has been cross-linked photochemically to three aminoacyl-tRNA synthetases, yeast phenylalanyl-tRNA synthetase, Escherichia coli isoleucyl-tRNA synthetase, and E. coli valyl-tRNA synthetase. The two non-cognate enzymes are known to interact with tRNA-Phe. In each complex, three regions on the tRNA are found to cross-link. Two of these are common to all of the complexes, while the third is unique to each. Thus, the cognate and non-cognate complexes bear considerable similarity to each other in the way in which the respective enzyme orients on tRNA-Phe, a result which was also established for the complexes of E. coli tRNA-Ile (BUDZIK, G.P., LAM, S.M., SCHOEMAKER, H.J.P., and SCHIMMEL, P.R. (1975) J. Biol. Chem. 250, 4433-4439). The common regions include a piece extending from the 5'-side of the acceptor stem to the beginning of the dihydrouridine helix, and a segment running from the 3' side of the extra loop into the TpsiC helix. These two regions overlap with and include some of the homologous bases found in eight tRNAs aminoacylated by yeast phenylalanyl-tRNA synthetase (ROE, B., SIROVER, M., and DUDOCK, B. (1973) Biochemistry 12, 4146-4153). Although well separated in the primary and secondary structure, these two segments are in close proximity in the crystallographic tertiary structure. In two of the complexes, the third cross-linked fragment is near to the two common ones. The picture which emerges is that the enzymes all interact with the general area in which the two helical branches of the L-shaped tertiary structure fuse together, with additional interactions on other parts of the tRNAas well.