Unmodified tRNA Research Articles

The turnover of tRNAs microinjected into animal cells

Red cell-mediated microinjection has been used to study tRNA turnover in SV3T3 mouse cells and TC7 cells, an African green monkey kidney line. The turnover of endogenous tRNA, measured by labeling with 3H-methionine, was first-order with half-lives of approximately one day in SV3T3 and two days in TC7 cells. 32PtRNA isolated from E. coli or TC7 cells turned over at the same rate as endogenous tRNA when injected into either SV3T3 or TC7 cells. This demonstrates that cellular processes, not properties inherent to tRNAs, are responsible for the difference in tRNA turnover observed between SV3T3 and TC7 cells. These results further indicate that the mechanism of tRNA turnover in mammaliam cells does not distinguish prokaryotic from eukaryotic tRNAs. In contrast to unmodified tRNA, glyoxalated tRNA was rapidly degraded upon injection. Thus altered tRNA's, like altered proteins, are turned over more rapidly in animal cells.

Aminoacylation and micleoside modification of in vitro synthesised transfer RNA

TRANSCRIPTION of transfer RNA (tRNA) genes in vitro by purified RNA polymerase has the advantage of producing completely unmodified tRNA precursors which provide a substrate for the study of the processes and modifications leading to the formation of mature tRNA molecules. We have shown1 that the tRNA genes, tRNA1Tyr (su3+ and su3−), tRNA2Gly su+A36, tRNA3Thr and tRNA2Tyr (carried by the respective ϕ80psu3+,− and λh80T phage DNA2,3), were transcribed in vitro by Escherichia coli RNA polymerase into polycistronic tRNA precursors. These precursors were cleaved on incubation with a supernatant fraction from E. coli, yielding mature size tRNA molecules1. The transcription in vitro of an active tRNA1Tyr su3+ by crude S-30 E. coli extracts or a combination of partially purified fractions has been described4–6. The formation of the primary transcription products of the tRNATyr gene was not, however, clearly demonstrated and it was concluded that an additional E. coli extract fraction would be required to enable purified RNA polymerase to function in tRNA synthesis5,6. We report here evidence indicating that the in vitro transcription of ϕ80psu3+,− or λh80T DNA by purified E. coli RNA polymerase alone produces tRNA precursor molecules which are not only processed to form mature-size tRNA1 but are also recognised by specific modifying enzymes. In addition the tRNA molecules (tRNA1Tyr, tRNA2Tyr, tRNA3Thr and tRNA2Gly) synthesised and processed in vitro, possess amino acid acceptance activity.

Isomeric phenylalanyl-tRNAs. Position of the aminoacyl moiety during protein biosynthesis.

The preparation of phenylalanyl-tRNA terminating in 3'-deoxyadenosine has been achieved by incubation of abbreviated tRNA (tRNA-CpC(OH)) with 3'-deoxyadenosine 5'-diphosphate and polynucleotide phosphorylase (EC 2.7.7.8), followed by aminoacylation. The isomeric phenylalanyl-tRNA terminating in 2'-deoxyadenosine was constructed by incubation of tRNA-CpC(OH) with 2'-deoxy-3'-O-phenylalanyladenosine 5'-diphosphate and polynucleotide phosphorylase. While tRNA is aminoacylated at the 2'-position, only the 3'-aminoacyl-tRNA is active as a peptide acceptor in the peptidyltransferase reaction. Both modified tRNAs were bound to the A-site as efficiently as unmodified tRNA, but neither was so efficient at P-site binding or as an acceptor in the peptidyltransferase reaction. Neither of the modified tRNAs acted as a donor in the peptidyltransferase reaction.

Recognition of altered [formula omitted] formylmethionine transfer RNA by bacterial T factor

Treatment of E. coli formylmethionine tRNA with sodium bisulfite produces six C → U base changes in the tRNA structure. Four of these modifications have no effect on the ability of tRNA f Met to be aminoacylated or formylated. Prior to bisulfite treatment, Met-tRNA f Met is not able to form a ternary complex with bacterial T factor and GTP, as measured by Sephadex G-50 gel filtration. After bisulfite treatment, a large portion of the modified tRNA is bound as T-GTP-Met-tRNA f Met. Formylation of bisulfite-modified Met-tRNA f Met completely eliminates T factor binding. Unmodified tRNA f Met is unique among the tRNAs sequenced to date in having a non-hydrogen-bonded base at the 5′ terminus. Bisulfite-catalyzed conversion of this unpaired C 1 to U 1 results in formation of a normal U 1-A 73 base pair at the end of the acceptor stem. It is likely that this structural alteration is responsible for the recognition of bisulfite-modified Met-tRNA f Met by T factor.

Preparation of a stable mercury derivative of tyrosine transfer RNA

Pentafluorophenylmercury chloride has been shown to react with both 4-thiouridine residues in tRNA Tyr to give a derivative with a covalent Hg-S bond. This tRNA derivative appears to be stable in solution under conditions used for crystallising and also for aminoacylating tRNA. Neither the derivative can be aminoacylated nor can unmodified tRNA Tyr be aminoacylated when in the presence of the tRNA-mercury derivative, suggesting that a stable complex is formed between the tRNA-mercury derivative and the amino acid synthetase.

Structure and function of Escherichia coli formylmethionine transfer RNA : II. Effect of modification of guanosine residues on aminoacyl synthetase recognition

Guanosine residues in Escherichia coli formylmethionine transfer RNA have been modified by photo-oxidation in the presence of methylene blue. After irradiation to the extent of 50% loss of amino-acid acceptor activity, separation of active and inactive molecules has shown that guanosine rsesidues in the stem adjacent to the dihydrouridine loop and the guanosine at position no. 2 from the 5′ terminus are not required for aminoacylation or transformylation. Active molecules containing these modifications are amino-acylated with a K m threefold higher than that for unmodified tRNA fMet. No change in V max occurs. Modification of a guanosine residue in the small loop joining the anticodon stem and the TΨC stem results in inactivation of methionine acceptor activity. This represents the first experimental evidence that a modification in this region affects the aminoacylation of a tRNA.