Transcription of Xenopus selenocysteine tRNA Ser (formerly designated opal suppressor phosphoserine tRNA) gene is directed by multiple 5'-extragenic regulatory elements.
A tRNA gene whose product is aminoacylated with serine and the serine moiety is then phosphorylated to form phosphoseryl-tRNA (see Hatfield, D. (1985) Trends Biochem. Sci. 10, 201-204 for review) has now been shown to form selenocysteyl-tRNA; hence the corresponding gene is designated as selenocysteine tRNA Ser (B. J. Lee, P. J. Worland, J. N. Davis, T. C. Stadtman, and D. Hatfield (1989) J. Biol. Chem. 264, in press). In the present study, we show that the expression of this unique tRNA gene is governed by at least three upstream regulatory elements. In initial studies, the relative efficiencies of transcription of the human, rabbit, chicken, and Xenopus selenocysteine tRNA genes were compared in vivo in Xenopus oocytes and in vitro in HeLa cell extracts. The Xenopus gene was severalfold more actively expressed, both in vivo and in vitro, than the human and rabbit genes, whereas the chicken gene was poorly expressed. Exchange of the 5'-flanking regions of the Xenopus and chicken genes, which have identical gene sequences, reversed their levels of transcription, demonstrating that a regulatory site or sites exist upstream of these genes. Deletion-substitution mutants in the Xenopus gene and its 5'-flanking sequence show in in vitro assays that 1) the level of transcription is reduced substantially when a GC-rich stretch that is immediately upstream of a TATA box in the -30 region is removed; 2) the level of transcription is virtually abolished when the TATA box is removed; and 3) deletions up to and further upstream of the GC-rich region do not affect the level of transcription. The same deletions, when used in in vivo assays, demonstrate a step-down in expression with the deletion removing the GC-rich region, a further step-down in expression with the deletion removing the TATA box, but the most pronounced reduction in expression was observed with a deletion removing an AT-rich region between nucleotides -62 and -76. Thus, a regulatory site was identified in vivo which was not detected in vitro, and transcription of the selenocysteine tRNA Ser gene is determined by multiple upstream regulatory elements.
Lee
B J
BJ
Laboratory of Experimental Carcinogenesis, National Cancer Institute, Bethesda, Maryland 20892.
Kang
S G
SG
Hatfield
D
D
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002645:Chickens; D002872:Chromosome Deletion; D003545:Cysteine / Q000031:analogs & derivatives* / Q000378:metabolism; D005796:Genes; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D012320:RNA Polymerase III; D012345:RNA, Transfer, Amino Acid-Specific / Q000235:genetics*; D012362:RNA, Transfer, Ser / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011817:Rabbits; D012045:Regulatory Sequences, Nucleic Acid; D012643:Selenium / Q000378:metabolism*; D017279:Selenocysteine; D014158:Transcription, Genetic; D014981:Xenopus / Q000235:genetics*
D012345:RNA, Transfer, Amino Acid-Specific; D012362:RNA, Transfer, Ser; D017279:Selenocysteine; D012320:RNA Polymerase III; D012643:Selenium; D003545:Cysteine