In some eucaryotes tRNA species occur containing exclusively uridine or ribosylthymine at position 54. In other eucaryotic tRNA species varying ratios of uridine to ribosylthymine have been found. To elucidate whether variation in the degree of U54 methylation in eucaryotic tRNA plays a functional role in protein synthesis, polysomal and overal cellular tRNAs in the lower eucaryote Dictyostelium discoideum were characterized during vegetative growth and the early developmental preaggregation stage.The nucleoside composition of the tRNAs was determined and U54-containing tRNA species were detected after methylation in vitro with the rT-forming enzyme from Escherichia coli. The following results were obtained. The results demonstrate that in D. discoideum, the major class tRNAs utilized for protein synthesis includes molecular species in which a Us4 residue has been methylated to rT. The ratio of rTs4 to Us4 in these tRNAs declines at a physiological stage at which the rate of protein synthesis is reduced. Since in vitro an eucaryotic U54-containing tRNA of this class translates mRNA less efficiently than its rT-containing counterpart [K. B. Marcu and B. S. Dudock (1976) Nature (Lond.) 261, 159-1621, it is assumed that these W4-containing tRNAs are rate-limiting in the elongation of peptide chains in eucaryotes.

Changes in the pattern of isoacceptors of tRNA Asn and alterations in modification of the guanine residue 34, the first position of the anticodon of tRNA Asn , have been observed in eukaryotes during differentiation. We use Dictyostelium discoideum as a model system to elucidate the possible involvement of tRNA Asn in developmental processes.Vegetative amoebae were induced to undergo developmental transition by nutrient starvation. Since amino acid starvation alone is a specific stimulus initiating development and unacylated tRNAs might be involved in control mechanisms of protein synthesis, the level of aminoacylation of tRNA Asn isoacceptors has been investigated.As early as two minutes after the onset of development, the aminoacylation of tRNA Asn specifically was reduced to about 30%, whereas at the same time 10 other tRNA species were found to be charged normally, i.e. to 70-100%. One of the two major isoacceptors, tRNA Asn , was completely deacylated, whereas the other one, tRNA Asn , accounted for the residual aminoacylation. Analyses of the modified nucleosides of highly purified tRNA Asn and tRNA Asn respectively, show that both isoacceptors are identical in their modification patterns except for the modification at the first position of the anticodon: tRNA Asn comprises queuine (Q),7-[(4,5-czs-dihydroxy-2-cyclopenten-1 -ylamino)methyl]-7-deazaguanine, whereas tRNA Asn contains guanine.

A genomic DNA fragment from Dictyostelium discoideum was characterized. This DNA, although 74% d(A + T)‐rich, codes for a putative tRNAValGUU. The tRNAVal gene overlaps at its 5′ half with another RNA polymerase III transcription unit. This RNA polymerase III transcription unit can be folded into a tRNA‐like shape and is comprised of significant amounts of invariant and semi‐invariant nucleotides present in all eukaryotic tRNAs. This unit contains the two promoter blocks defined for RNA polymerase III, which are homologous to recently defined promoter elements to the extent of 76–88% (A block) and 86–93% (B block) respectively [Sharp et al. (1981) Proc. Natl Acad. Sci. USA 78, 6657–6661]. Both of the overlapping class III genes are transcribed in germinal vesicle extracts prepared from Xenopus laevis oocytes as a single transcription unit, resulting in an unusually large product compared to primary transcripts of other tRNA genes. The unit is not transcribed in HeLa extracts but it competes very strongly for transcription factor(s) under the conditions of stable transcription complex formation. Although the whole unit is transcribed, it is believed that only one functional product is formed. Therefore we define the tRNA‐like structure, coded for on this class III transcription unit, as a putative tRNA ‘pseudogene’ meaning that, although it is transcribed by RNA polymerase III, it is not likely to mature to a functional tRNA.

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