trans-Diamminedichloroplatinum(II), a reversible RNA-protein cross-linking agent. Application to the ribosome and to an aminoacyl-tRNA synthetase/tRNA complex

A new approach allowing detection of contact points between RNAs and proteins has been developed using trans-diamminedichloroplatinum(II) as the cross-linking reagent. The advantage of the method relies on the fact that the coordination bonds between platinum and the potential acceptors on proteins and nucleic acids (mainly S of cysteine or methionine residues; N of imidazole rings in histidine residues; N7 of guanine, N1 of adenine, and N3 of cytosine residues) can be reversed, so that the cross-linked oligonucleotides or peptides in contact within a complex can be analyzed directly. The method was worked out with the ribosome from Escherichia coli and the tRNAVal/valyl-tRNA synthetase system from the yeast Saccharomyces cerevisiae. In the first system the platinum approach permitted detection of ribosomal proteins cross-linked to 16S rRNA within the 30S subunits (mainly S18 and to a lower extent S3, S4, S11, and S13/S14); in the second system major oligonucleotides of tRNAVal cross-linked to valyl-tRNA synthetase were detected in the anticodon stem and loop, in the variable loop, and in the 3' terminal amino acid accepting region. These results are discussed in light of the current knowledge on ribosome and tRNAs and of potential applications of the methodology.     

Dinucleoside tetraphosphate variations in cultured tumor cells during their cell cycle and growth

Asynchronous and synchronized cultures of A549 and HTC cells were used to detect possible, cell cycle or cell density specific variations in the intracellular pools of dinucleoside tetraphosphates (Ap4X). No important variations of the nucleotide pools were observed during cell growth. When HTC cells were released from mitotic arrest, a decrease by a factor of N3 Ap4X and ATP levels was observed when the cells entered the G1 phase. This decrease is essentially due to cell doubling. When A549 cells were released from an arrest at the G1/S boundary, the nucleotide pool size increased slightly during the G2 phase just before mitosis. This result is in agreement with both earlier data from our laboratory and the observed decrease in Ap4X pool after release from mitotic-arrested HTC cells. These results suggest that the Ap4X and ATP pools are only subjected to very small variations during the cell cycle, essentially in the G2 phase and after mitosis.     

Crosslinking of elongation factor Tu to tRNA(Phe) by trans-diamminedichloroplatinum (II). Characterization of two crosslinking sites on EF-Tu

In a preceding paper [(1987) Nucleic Acids Res. 15, 5787-5801], we have used trans-diamminedichloroplatinum (II) to induce reversible RNA-protein crosslinks within the ternary EF-Tu/GTP/Phe-tRNA(Phe) complex and have identified two crosslinking sites on the tRNA. The aim of the present paper is to determine the crosslinking sites on EF-Tu. Two tryptic peptides located in domain I could be identified, a major one (residues 45-74) and a minor one (residues 117-154). The use of Staphylococcus aureus V8 protease led to the isolation of two major peptides (residues 56-68 and 64-68) and one minor peptide (118-124). These results are discussed in the light of the current knowledge of the topography of the EF-Tu/tRNA complex.     

Identification of the major tRNA(Phe) binding domain in the tetrameric structure of cytoplasmic phenylalanyl-tRNA synthetase from baker's yeast

Native cytoplasmic phenylalanyl-tRNA synthetase from baker's yeast is a tetramer of the alpha 2 beta 2 type. On mild tryptic cleavage it gives rise to a modified alpha 2 beta 2 form that has lost the tRNA(Phe) binding capacity but is still able to activate phenylalanine. In this paper are presented data concerning peptides released by this limited proteolytic conversion as well as those arising from exhaustive tryptic digestion of the truncated beta subunit. Each purified peptide was unambiguously assigned to a unique stretch of the beta subunit amino acid sequence that was recently determined via gene cloning and DNA sequencing. Together with earlier results from affinity labelling studies the present data show that the Lys 172-Ile 173 bond is the unique target of trypsin under mild conditions and that the N-terminal domain of each beta subunit (residues 1-172) contains the major tRNA(Phe) binding sites.     

Is Ap4A involved in DNA repair processes?

Hepatoma tissue culture (HTC) cells were incubated in the presence of the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) to study the variations in the bisnucleosides polyphosphates (Ap4X) pool size. A transient but sensitive accumulation of these compounds is observed; if 3-aminobenzamide (3AB) which is a potent inhibitor of the ADP-ribosyltransferase (ADPRT) is added after the MNNG treatment, a more pronounced and persistent accumulation of Ap4X can be seen. A moderate heat-shock (30 min at 43 degrees C) results also in a small accumulation of Ap4X but the shape of the accumulation curve is quite different and the increase of the Ap4X pool is not sensitive to the presence of 3AB. However, both MNNG treatment and hyperthermia cause a marked inhibition of protein synthesis. On the other hand, the ADPRT activity is enhanced in the presence of MNNG whereas hyperthermia has little or a slightly inhibitory effect on this activity. These results suggest that MNNG treatment triggers an Ap4X accumulation in eukaryotic cells different from that observed after heat-shock and it seems likely that these compounds are involved in the DNA excision repair system in which the ADPRT enzyme is also implicated.     

Lack of correlation between extensive accumulation of bisnucleoside polyphosphates and the heat-shock response in eukaryotic cells

The accumulation in large amounts of bisnucleoside polyphosphates (Ap4X) after heat shock in Xenopus laevis oocytes or cultured hepatoma cells (HTC cells) is observed after exposure to temperatures of 45 degrees C or higher. The accumulation is a transient phenomenon, with the collapse in cellular ATP concentration severely affecting the rate of synthesis of Ap4X, allowing degrading activities to empty the pool of these compounds under prolonged heat shock. This accumulation of Ap4X to high levels, compared to the basic content, is only observed under conditions leading to irreversible damage, ultimately resulting in the death of the cell. It is shown that the increase in Ap4X after hyperthermia is due to the partial or almost complete inhibition of their degradation pathways, rather than to a stimulation of their rate of synthesis. Finally, the synthesis of heat-shock proteins could be observed under conditions which do not lead to important accumulation of Ap4X, therefore ruling out the possibility that these adenylylated nucleotides would behave as chemical signals ("alarmones") triggering the synthesis of heat-shock proteins. Nevertheless, on the basis of our earlier results (Guédon, G., Sovia, D., Ebel, J. P., Befort, D., and Remy, P. (1985) Embo J. 4, 3743-3749), it cannot be excluded that Ap4X might play a role in the regulation of the heat-shock response; this would, however, rely on variations in Ap4X concentrations which do not exceed a factor of 2.