Lee B J, Kang S G, Hatfield D
Laboratory of Experimental Carcinogenesis, National Cancer Institute, Bethesda, Maryland 20892.
J Biol Chem. 1989 Jun 5;264(16):9696-702.
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.
一个其产物被丝氨酸氨酰化，随后丝氨酸部分被磷酸化形成磷酸丝氨酰 - tRNA的tRNA基因（见哈特菲尔德，D.（1985年）《生物化学趋势》10，201 - 204的综述），现已证明它能形成硒代胱氨酰 - tRNA；因此相应的基因被命名为硒代半胱氨酸tRNA Ser（B. J. 李、P. J. 沃兰、J. N. 戴维斯、T. C. 斯塔特曼和D. 哈特菲尔德（1989年）《生物化学杂志》264，即将发表）。在本研究中，我们表明这个独特的tRNA基因的表达受至少三个上游调控元件的控制。在初步研究中，比较了人、兔、鸡和非洲爪蟾硒代半胱氨酸tRNA基因在非洲爪蟾卵母细胞体内以及在HeLa细胞提取物中的转录相对效率。非洲爪蟾基因在体内和体外的表达活性都比人和兔的基因高几倍，而鸡的基因表达较差。具有相同基因序列的非洲爪蟾和鸡基因的5'侧翼区域的交换，逆转了它们的转录水平，表明在这些基因的上游存在一个或多个调控位点。非洲爪蟾基因及其5'侧翼序列的缺失 - 替换突变体在体外试验中表明：1）当 - 30区域中TATA框上游紧邻的富含GC的片段被去除时，转录水平大幅降低；2）当TATA框被去除时，转录水平几乎完全消失；3）在富含GC区域及其上游的缺失并不影响转录水平。当在体内试验中使用相同的缺失时，随着去除富含GC区域的缺失，表达呈逐步下降，随着去除TATA框的缺失，表达进一步下降，但在去除核苷酸 - 62至 - 76之间富含AT区域的缺失时，观察到表达下降最为明显。因此，在体内鉴定出了一个在体外未检测到的调控位点，并且硒代半胱氨酸tRNA Ser基因的转录由多个上游调控元件决定。
