Opal suppressor serine tRNAs from bovine liver form phosphoseryl-tRNA.

Abstract

An unusual minor species of bovine liver serine tRNA has previously been isolated, sequenced, and found to suppress the UGA termination codon in protein synthesis in vitro [Diamond, A., Dudock, B. & Hatfield, D. (1981) CeU25, 497-506]. We have now found that this tRNA can be a substrate in a specific phosphorylation reaction in which phosphoseryl-tRNA is formed. Moreover, bovine liver contains a second UGA suppressor serine tRNA (tRNASerA; N is a modified nucleoside) which also forms phosphoseryl-tRNA. The nucleotide sequence and coding properties of tRNASeA are presented.The suppression of termination codons by naturally occurring suppressor tRNAs has been best studied in prokaryotic systems. The genomes ofboth bacteriophage Q(3 (1, 2) and A (3) code for proteins that arise as a consequence of partial readthrough of UGA termination signals by the normal tryptophan tRNA ofthe host. In eukaryotes, tRNAs that suppress nonsense codons have been observed in yeast and Drosophila (4) as well as in mammalian cells (5, 6). A tyrosine tRNA from Drosophila that lacks the highly modified Q base in its anticodon is responsible for suppressing the termination codon UAG, whereas the corresponding tyrosine tRNA with a fully modified Q base does not read UAG (4). In mammalian cells a naturally occurring suppressor tryptophan tRNA has been shown to read the UGA stop codon of -hemoglobin mRNA both in rabbit reticulocyte lysates and in intact reticulocytes (5). In addition, we have reported the characterization and nucleotide sequence of an unusual bovine liver UGA suppressor serine tRNA (tRNACrA) (6).In this paper we report that tRNASCA can be a substrate in a specific phosphorylation reaction in which phosphoseryltRNA is formed. In addition, there exists another bovine liver suppressor serine tRNA (tRNASerA in which N is a modified nucleoside whose structure has not yet been determined) which also forms phosphoseryl-tRNA. The nucleotide sequence and coding properties of tRNAeA are presented here. This tRNA, like tRNACerA is aminoacylated with serine and suppresses the UGA termination codon in vitro. The nucleotide sequences of tRNAASer and tRNACerA differ in only six positions, all located in the 5' 40% of the molecule.MATERIALS AND METHODS tRNA was isolated from bovine liver (7), estrogen-treated rooster liver (8), and rabbit reticulocytes (7). Aminoacyl-tRNA synthetases were prepared from rabbit reticulocytes as described (7). Conditions of aminoacylation were the same as those described (6, 7) except that [3H]serine (14-28 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels) was used. Bovine liver tRNAeCA was purified as described (6). tRNASA was isolated from fresh bovine liver (9) and subsequently purified by three successive chromatographic runs on RPC-5 columns, first in a linear 0.525-0.725 M NaCl gradient as described (7), followed by the same NaCl gradient run in the absence of Mg2", and finally in a 0.45 M NaCl (150 ml) to 1.0 M NaCl (75 ml) concave gradient. The final purification was by preparative electrophoresis on a ...

Identification and characterization of phosphoseryl-tRNA [Ser]Sec kinase

“…Since this original report, the identity, isolation, and cellular role of PSTK, as well as phosphoseryltRNA [Ser]Sec , have remained largely obscure. The minor phosphoseryl-tRNA and another minor seryl-tRNA identified in bovine, rabbit, and chicken livers that decoded the stop codon UGA (12) were shown to be Sec tRNA [Ser]Sec (13). It was subsequently reported that phosphoseryl-tRNA [Ser]Sec was the intermediate in the biosynthesis of Sec in mammals (21,22) and in bacteria (23).…”

“…For example, in 1970, a minor seryl-tRNA was reported to form phosphoseryl-tRNA by a kinase activity from rooster liver (11), and a minor seryl-tRNA from bovine, rabbit, and chicken livers was reported to specifically decode the nonsense codon UGA (12). Although it was subsequently shown that both the minor seryl-tRNA that formed phosphoseryl-tRNA and the one that decoded UGA were the same tRNA (13), it was not known at the time these components were discovered that they played instrumental roles in the biosynthesis of Sec and its incorporation into protein as the 21st amino acid in the genetic code. The minor serine tRNA was subsequently found to be selenocysteyl-tRNA [Ser]Sec (10), but the role of the putative kinase that phosphorylated seryltRNA [Ser]Sec has not been resolved.…”

“…13. The phosphorylated adenosine product of the kinase reaction was identified by using [␣-32 P]ATP and analyzing the 32 P-labeled product by TLC.…”

Purification and properties of suppressor seryl‐tRNA:ATP phosphotransferase from bovine liver

“…These two tRNAsSeT were identical [3] and the primary structure of this tRNA from bovine liver [4] and the tRNA gene structure of chicken were determined [5]. The tRNA plays a suppressor role in in vitro globin synthesis [3], but it is unclear whether phosphoserine on the tRNA is incorporated in a polypeptide chain of protein synthesis. cry1 S-300 and affinity chromatography on Blue Sepharose, as one band on disc gels.…”

New Developments in Selenium Biochemistry: Selenocysteine Biosynthesis in Eukaryotes and Archaea

“…For example, a kinase that phosphorylated a minor seryltRNA to form O-phosphoseryl-tRNA was reported in rooster liver as early as 1970 [15]. This same year a minor seryl-tRNA from mammalian and chicken liver was found to decode specifically the termination codon, UGA [16], which was subsequently shown to be the same tRNA that formed phosphoseryl-tRNA [17]. This tRNA was subsequently identified as Sec tRNA [Ser]Sec [6].…”