Kim D, Johnson J
Department of Molecular Biology, University of Wyoming, Laramie 82071.
J Biol Chem. 1988 May 25;263(15):7316-21.
The number of different tRNA species in Saccharomyces cerevisiae known to be capable of suppressing termination of translation at UAG, UAA, and UGA codons is limited to those which insert tyrosine, leucine, and serine. Suppressor tRNAs that insert other amino acids, even those whose anticodons differ from the expected recognition sequences for nonsense codons by a single nucleotide, have never been identified via classical genetic analysis. We have used site-directed mutagenesis to convert the anticodon of a cloned tRNATrp gene from CCA to CTA with the expectation that this gene would produce tRNA molecules capable of interacting with the UAG terminator codon. We show that this form of the gene can be transcribed and spliced in vitro to produce mature tRNA with the expected base sequence. The putative suppressor gene has been introduced into several S. cerevisiae host strains using the centromere vector YCp19. Efficient suppression of amber mutations met8-1, tyr7-1, and lys2-801 results from the presence of the CTA form of tDNATrp. Two UAA mutants, leu2-1 and ade2-101, and the UGA marker his4-260 are not suppressed.
已知能够抑制酿酒酵母中UAG、UAA和UGA密码子处翻译终止的不同tRNA种类仅限于那些插入酪氨酸、亮氨酸和丝氨酸的tRNA。即使那些反密码子与无义密码子的预期识别序列仅相差一个核苷酸的插入其他氨基酸的抑制性tRNA，也从未通过经典遗传分析鉴定出来。我们利用定点诱变将克隆的tRNATrp基因的反密码子从CCA转换为CTA，期望该基因能产生能够与UAG终止密码子相互作用的tRNA分子。我们表明这种形式的基因可以在体外转录和剪接，以产生具有预期碱基序列的成熟tRNA。使用着丝粒载体YCp19已将推定的抑制基因导入几种酿酒酵母宿主菌株。tDNATrp的CTA形式的存在导致对琥珀突变met8-1、tyr7-1和lys2-801的有效抑制。两个UAA突变体leu2-1和ade2-101以及UGA标记his4-260未被抑制。
