The long extra arms of human tRNA((Ser)Sec) and tRNA(Ser) function as major identify elements for serylation in an orientation-dependent, but not sequence-specific manner.

Selenocysteine tRNA [tRNA((Ser)Sec)] is charged with serine by the same seryl-tRNA synthetase (SerRS) as the canonical serine tRNAs. Using site-directed mutagenesis, we have introduced a series of mutations into human tRNA((Ser)Sec) and tRNA(Ser) in order to study the identity elements of tRNA((Ser)Sec) for serylation and the effect of the orientation of the extra arm. Our results show that the long extra arm is one of the major identity elements for both tRNA(Ser) and tRNA((Ser)Sec) and gel retardation assays reveal that it appears to be a prerequisite for binding to the cognate synthetase. The long extra arm functions in an orientation-dependent, but not in a sequence-specific manner. The discriminator base G73 is another important identity element of tRNA((Ser)Sec), whereas the T- and D-arms play a minor role for the serylation efficiency.     

Flexible zinc finger requirement for binding of the transcriptional activator staf to U6 small nuclear RNA and tRNA(Sec) promoters.

The transactivator Staf, which contains seven zinc finger motifs, exerts its effect on gene expression by binding to specific targets in small nuclear RNA (snRNA) and snRNA-type gene promoters. In this work, binding site selection allowed us to identify the 21-base pair ATTACCCATAATGCATYGCGG sequence as the high affinity consensus binding site for Staf. It shows a high sequence divergence with Staf-responsive elements in the Xenopus selenocysteine tRNA (tRNA(Sec)) and human U6 snRNA promoters. By using a combination of approaches, we analyzed the interaction of wild-type and truncated Staf zinc finger domains with the consensus, Xenopus tRNA(Sec), and human U6 sites. Two main conclusions emerged from our data. First, the data clearly indicate that zinc finger 7 does not establish base-specific contacts in Staf-DNA complexes. The second conclusion concerns zinc finger 1, which is required for the binding to the Xenopus tRNA(Sec) site but is dispensable in the case of the human U6 site. Taking into account the sequence differences in the two sites, these findings demonstrate that Staf utilizes zinc finger 1 in a rather flexible manner, illustrating how a protein can interact with DNAs containing targets of different sequences.     

Selenocysteine tRNA[Ser]Sec gene is ubiquitous within the animal kingdom.

Recently, a mammalian tRNA which was previously designated as an opal suppressor seryl-tRNA and phosphoseryl-tRNA was shown to be a selenocysteyl-tRNA (B. J. Lee, P. J. Worland, J. N. Davis, T. C. Stadtman, and D. Hatfield, J. Biol. Chem. 264:9724-9727, 1989). Hence, this tRNA is now designated as selenocysteyl-tRNA[Ser]Sec, and its function is twofold, to serve as (i) a carrier molecule upon which selenocysteine is biosynthesized and (ii) as a donor of selenocysteine, which is the 21st naturally occurring amino acid of protein, to the nascent polypeptide chain in response to specific UGA codons. In the present study, the selenocysteine tRNA gene was sequenced from Xenopus laevis, Drosophila melanogaster, and Caenorhabditis elegans. The tRNA product of this gene was also identified within the seryl-tRNA population of a number of higher and lower animals, and the human tRNA[Ser]Sec gene was used as a probe to identify homologous sequences within genomic DNAs of organisms throughout the animal kingdom. The studies showed that the tRNA[Ser]Sec gene has undergone evolutionary change and that it is ubiquitous in the animal kingdom. Further, we conclude that selenocysteine-containing proteins, as well as the use of UGA as a codon for selenocysteine, are far more widespread in nature than previously thought.     

Antideterminants present in minihelix(Sec) hinder its recognition by prokaryotic elongation factor Tu.

During protein biosynthesis, all aminoacylated elongator tRNAs except selenocysteine-inserting tRNA Sec form ternary complexes with activated elongation factor. tRNA Sec is bound by its own translation factor, an elongation factor analogue, e.g. the SELB factor in prokaryotes. An apparent reason for this discrimination could be related to the unusual length of tRNA Sec amino acid-acceptor branch formed by 13 bp. However, it has been recently shown that an aspartylated minihelix of 13 bp derived from yeast tRNA Asp is an efficient substrate for Thermus thermophilus EF-Tu-GTP, suggesting that features other than the length of tRNA Sec prevent its recognition by EF-Tu-GTP. A stepwise mutational analysis of a minihelix derived from tRNA Sec in which sequence elements of tRNA Asp were introduced showed that the sequence of the amino acid- acceptor branch of Escherichia coli tRNA Sec contains a specific structural element that hinders its binding to T.thermophilus EF-Tu-GTP. This antideterminant is located in the 8th, 9th and 10th bp in the acceptor branch of tRNA Sec, corresponding to the last base pair in the amino acid acceptor stem and the two first pairs in the T-stem. The function of this C7.G66/G49.U65/C50.G64 box was tested by its transplantation into a minihelix derived from tRNA Asp, abolishing its recognition by EF-Tu-GTP. The specific role of this nucleotide combination is further supported by its absence in all known prokaryotic elongator tRNAs.