Ability of modified forms of phenylalanine tRNA to stimulate guanosine pentaphosphate synthesis by the stringent factor-ribosome complex of E. coli

Abstract

PRNhe 4 tRNAPhe of E. coli, modified at its 4-thiouridine ( Srd) and 3-(3-amino-3-carboxypropyl)uridine (nbt3UXd) residues, was tested for its ability to induce (p)ppGpp synthesis. The Srd residue was derivatized with the p-azidophenacyl group, cross-linked to Cyd13, and the borohydride reduction'product of the cross-link was prepared. The nbt3Urd residue was derivatized with the N-(4-azido-2-nitrophenyl)glycyl group. None of these derivatives had more than a minor effect on the affinity of the tRNA for the stringent factorribosome complex, and no effect at all on the maximum velocity of (p)ppGpp synthesis, either at 2 or 82 mM NH4C1. These two regions of the tRNA which are on opposite faces of the tRNA molecule do not appear to be structurally important for recognition by the stringent factor-ribosome complex. They may provide useful sites, therefore, for the introduction of photoaffinity or fluorescent probes with which to study tRNA-stringent factor recognition.

Participation of X47-fluorescamine modified E. coli tRNAs in in vitro protein biosynthesis

“…The fluorescamine modification on the variable loop of the aminoacyl-tRNA does not influence its interaction with the ribosomal A-site. The results obtained with tRNA -C-C-A(XF47) support the recent data obtained with tRNA species modified on the X47 residue; such tRNAs were active in the induction of the ribosome dependent synthesis of ppGpp (magic spots) (10), which is known to require the binding of tRNA to the ribosomal A-site.…”

“…The incorporation of a spectroscopic label into the variable loop of various tRNAs results in differing effects on the biological activity of the nucleic acid. Acylation of the minor nucleoside X, 3-N(3-amino-3-carboxypro-C Information Retrieval Limited I Falconberg Court London Wl V 5FG England pyl)uridine, (7) leads to complete (8,9), partial (10) or no loss (11)(12)(13)(14) of of the acceptor activity of the modified tRNAs. In fact, there is no satisfactory explanation available whether the inactivation of X-base modified tRNA is associated with the participation of the variable loop of tRNA in the interaction with the cognate aminoacyl-tRNA synthetase or if this effect is due to unspecific side reactions or irreversible inactivation of the tRNA during the modification procedures.…”

Position of transfer ribonucleic acid on Escherichia coli ribosomes. Distance from the 3' end of 16S ribonucleic acid to three points on phenylalanine-accepting transfer ribonucleic acid in the donor site of 70S ribosomes

“…Phe-tRNAxLR was aminoacylated by using high Mg2+-Me2SO conditions, since cross-linking, or any other modification of Srd8 in E. coli tRNAPhe, interferes with the acylation activity of E. coli synthetase (Ofengand & Liou, 1978). The conditions were 25 mM Tris, pH 8.5, 0.5 mM ATP, 40 mM Mg2+, 20% Me2SO, 1 mM dithiothreitol, 20 µ phenylalanine, and 2 A260 units/mL IRNA^r and partially-purified synthetase at 30 °C.…”