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Tif6p (eIF6) is necessary for 60S biogenesis, rRNA maturation and must be released from 60S to permit 80S assembly and translation. We characterized Tif6p interactors. Tif6p is mostly on 66S-60S pre-ribosomes, partly free. Tif6p complex(es) contain nucleo-ribosomal factors and Asc1p. Surprisingly, Tif6p particle contains the low-abundance endonuclease Sen34p. We analyzed Sen34p role on rRNA/tRNA synthesis, in vivo. Sen34p depletion impairs tRNA splicing and causes unexpected 80S accumulation. Accordingly, Sen34p overexpression causes 80S decrease and increased polysomes which suggest increased translational efficiency. With delayed kinetics, Sen34p depletion impairs rRNA processing. We conclude that Sen34p is absolutely required for tRNA splicing and that it is a rate-limiting element for efficient translation. Finally, we confirm that Tif6p accompanies 27S pre-rRNA maturation to 25S rRNA and we suggest that Sen34p endonuclease in Tif6p complex may affect also rRNA maturation.

To study tRNA import into plant mitochondria, we have set up a system to follow the fate in vivo of tRNA transcripts introduced into plant protoplasts by electroporation. Conditions were optimized for maximum tRNA uptake into potato protoplasts. We have shown that in vitro synthesized tRNA transcripts are poor substrates due to rapid degradation leading to low efficiencies of transfer and short life in protoplasts. Labelled natural tRNAs were more efficiently electroporated into protoplasts and they remained stable during protoplast culture. We have observed import into mitochondria of total and purified cytosolic tRNAs in protoplasts but the process was not specific for the tRNA species which are normally imported.

The in vivo incorporation of the photoactivable uridine analogue 4-thiouridine into the RNAs of an Escherichia coli K12 pyrD strain has been optimised. s4Urd uptake in RNAs appears to be strikingly dependent upon the age of the preculture, i.e. the number of generations the cells have undergone immediately before dilution in the thiolation medium. Conditions have been set up where efficient RNA thiolation occurs in cells growing exponentially at 50 to 70% the rate of the control. The substitution level s4U/U is maximal after growth for 9 to 10 generations in the thiolation medium and reaches 17 +/- 3% in tRNA and bulk RNA. Most of ribosomal derived ribonucleoproteins, 65 +/- 5%, sediment as 70S ribosomes (s4U/U = 7 +/- 2%) on a high Mg2+ sucrose gradient. The thiolated RNAs were characterized by their migration on a thiol-specific affinity electrophoretic gel.

Explants of mouse mammary glands were cultured with and without prolactin in the presence of inorganic [32P] to estimate the effect of prolactin on tRNA synthesis. Labeled tRNA was extracted and characterized by two-dimensional gel electrophoresis. tRNA synthesis was 2-3 fold greater in the presence of prolactin; and the synthesis rate of each resolvable tRNA species was increased proportionally. tRNA populations from mouse mammary tissues at three stages of development were also examined. Alterations were noted between early pregnant and fully lactating tissues. The results of this study provide evidence that the tRNA population, which is known to be "specialized" for casein synthesis in the mammary gland, is determined as the gland develops and prepares for lactation.

The transcription of a mouse genomic segment containing four tRNA genes, coding for a tRNA(Ala), a tRNA(Ile), a tRNA(Pro) and a tRNA(Lys), has been studied in a HeLa cell extract, demonstrating that differences among their transcriptional efficiencies are evident using as templates either the natural cluster or an equimolecular mixture of the four isolated genes. Nevertheless, the structure of the cluster influences the transcriptional efficiency of the clustered genes. In fact, a cis-acting inhibitory sequence has been located at about 400 bp downstream of the tRNA(Pro) coding sequence. Moreover rearrangements of the reciprocal position of the various tRNA genes within the cluster results in significant changes in the transcriptional rates of the individual transcriptional units.