INVITRO SYNTHESIS OF SELENOCYSTEINYL-TRANSFER RNAUCA FROM SERYL-TRANSFER RNAUCA - INVOLVEMENT AND CHARACTERIZATION OF THE SELD GENE-PRODUCT
INVITRO SYNTHESIS OF SELENOCYSTEINYL-TRANSFER RNAUCA FROM SERYL-TRANSFER RNAUCA - INVOLVEMENT AND CHARACTERIZATION OF THE SELD GENE-PRODUCT
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DOI：
10.1073/pnas.87.2.543
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发表时间：
1990-01-01
影响因子：
11.1
通讯作者：
BOCK, A
中科院分区：
文献类型：
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作者：
LEINFELDER, W;FORCHHAMMER, K;BOCK, A
从大肠杆菌中克隆了与硒代谢有关的seld基因，并对其进行了序列分析。SELD编码一个由347个氨基酸组成的蛋白质，计算出的相对分子质量为36,687。通过在噬菌体T7启动子/聚合酶系统中表达seld基因产物的分析，证实了预测的蛋白质的相对分子质量。基因阻断实验表明，SELD蛋白既是将硒掺入tRNA的修饰核苷5-甲氨基甲基-2-硒尿苷中所必需的，也是从与tRNAUCASer连接的L丝氨酸残基生物合成硒半胱氨酸所必需的。TRNAUCASer被纯化，用L-丝氨酸进行氨基酰化，并作为底物用于建立硒半胱氨酸生物合成的体外体系。通过使用SELD和SELA基因产物均高产量的提取物，实现了硒半胱氨酸tRNAUCASer的高效形成。结果表明，硒半胱氨酸是由L丝氨酸与tRNAUCA结合合成的，与作为还原型硒供体的SELD是一致的。
The selD gene from Escherichia coli, whose product is involved in selenium metabolism, has been cloned and sequenced. selD codes for a protein of 347 amino acids with a calculated molecular weight of 36,687. Analysis of the selD gene product through expression of the gene in the phage T7 promoter/polymerase system confirmed the predicted molecular weight of the protein. Gene disruption experiments demonstrated that the SelD protein is required both for the incorporation of selenium into the modified nucleoside 5-methylaminomethyl-2-selenouridine of tRNA and for the biosynthesis of selenocysteine from an L-serine residue ester-bonded to tRNAUCASer. tRNAUCASer has been purified, aminoacylated with L-serine, and used as a substrate for the development of an in vitro system for selenocysteine biosynthesis. Efficient formation of selenocysteinyl-tRNAUCASer was achieved by using extracts in which both the selD and the selA gene products were overproduced. The results demonstrate that selenocysteine is synthesized from L-serine bound to tRNAUCA and they are in accord with SelD functioning as a donor of reduced selenium.