Johansson Marcus J O, Byström Anders S
Department of Molecular Biology, Umeå University, 901 87 Umeå, Sweden.
RNA. 2004 Apr;10(4):712-9. doi: 10.1261/rna.5198204.
The biogenesis of transfer RNA is a process that requires many different factors. In this study, we describe a genetic screen aimed to identify gene products participating in this process. By screening for mutations lethal in combination with a sup61-T47:2C allele, coding for a mutant form of, the nonessential TAN1 gene was identified. We show that the TAN1 gene product is required for formation of the modified nucleoside N(4)-acetylcytidine (ac(4)C) in tRNA. In Saccharomyces cerevisiae, ac(4)C is present at position 12 in tRNAs specific for leucine and serine as well as in 18S ribosomal RNA. Analysis of RNA isolated from a tan1-null mutant revealed that ac(4)C was absent in tRNA, but not rRNA. Although no tRNA acetyltransferase activity by a GST-Tan1 fusion protein was detected, a gel-shift assay revealed that Tan1p binds tRNA, suggesting a direct role in synthesis of ac(4)C(12). The absence of the TAN1 gene in the sup61-T47:2C mutant caused a decreased level of mature, indicating that ac(4)C(12) and/or Tan1p is important for tRNA stability.
转运RNA的生物合成是一个需要多种不同因子的过程。在本研究中,我们描述了一项旨在鉴定参与该过程的基因产物的遗传筛选。通过筛选与编码非必需TAN1基因的突变形式的sup61-T47:2C等位基因组合时致死的突变,鉴定出了TAN1基因。我们表明,TAN1基因产物是tRNA中修饰核苷N(4)-乙酰胞苷(ac(4)C)形成所必需的。在酿酒酵母中,ac(4)C存在于亮氨酸和丝氨酸特异性tRNA的第12位以及18S核糖体RNA中。对从tan1缺失突变体中分离的RNA的分析表明,tRNA中不存在ac(4)C,但rRNA中存在。尽管未检测到GST-Tan1融合蛋白的tRNA乙酰转移酶活性,但凝胶迁移试验表明Tan1p结合tRNA,提示其在ac(4)C(12)合成中起直接作用。sup61-T47:2C突变体中TAN1基因的缺失导致成熟水平降低,表明ac(�C(12)和/或Tan1p对tRNA稳定性很重要。