King M P, Attardi G
Division of Biology, California Institute of Technology, Pasadena 91125.
J Biol Chem. 1993 May 15;268(14):10228-37.
In human mitochondrial DNA (mtDNA), the tRNA genes are located in three different transcription units that are transcribed at three different rates. To analyze the regulation of tRNA formation by the three transcription units, we have examined the steady-state levels and metabolic properties of the tRNAs of HeLa cell mitochondria. DNA excess hybridization experiments utilizing separated strands of mtDNA and purified tRNA samples from exponential cells long term labeled with [32P]orthophosphate have revealed a steady-state level of 6 x 10(5) tRNA molecules/cell, with three-fourths being encoded in the H-strand and one-fourth in the L-strand. Hybridization of the tRNAs with a panel of M13 clones of human mtDNA containing, in most cases, single tRNA genes and a quantitation of two-dimensional electrophoretic fractionations of the tRNAs have shown that the steady-state levels of tRNA(Phe) and tRNA(Val) are two to three times higher than the average level of the other H-strand-encoded tRNAs and three to four times higher than the average level of the L-strand-encoded tRNAs. Similar experiments carried out with tRNAs isolated from cells labeled with very short pulses of [5-3H]uridine have indicated that the rates of formation of the individual tRNA species are proportional to their steady-state amounts. Therefore, the approximately 25-fold higher rate of transcription of the tRNA(Phe) and tRNA(Val) genes relative to the other H-strand tRNA genes and the 10-16-fold higher rate of transcription of the L-strand tRNA genes relative to the H-strand tRNA genes are not reflected in the steady-state levels or the rates of formation of the corresponding tRNAs. A comparison of the steady-state levels of the individual tRNAs with the corresponding codon usage for protein synthesis, as determined from the DNA sequence and the rates of synthesis of the various polypeptides, has not revealed any significant correlation between the two parameters.
在人类线粒体DNA（mtDNA）中，tRNA基因位于三个不同的转录单元，以三种不同的速率进行转录。为了分析这三个转录单元对tRNA形成的调控作用，我们检测了HeLa细胞线粒体tRNA的稳态水平和代谢特性。利用从指数生长期细胞中分离出的mtDNA单链以及用[32P]正磷酸盐长期标记的纯化tRNA样本进行的DNA过量杂交实验表明，每个细胞中tRNA分子的稳态水平为6×10⁵，其中四分之三由重链（H链）编码，四分之一由轻链（L链）编码。tRNA与一组包含大多数情况下单个tRNA基因的人类mtDNA的M13克隆进行杂交，并对tRNA的二维电泳分离进行定量分析，结果显示tRNA（Phe）和tRNA（Val）的稳态水平比其他由H链编码的tRNA平均水平高两到三倍，比由L链编码的tRNA平均水平高三到四倍。用从用[5-³H]尿苷极短脉冲标记的细胞中分离出的tRNA进行的类似实验表明，各个tRNA种类的形成速率与其稳态量成正比。因此，tRNA（Phe）和tRNA（Val）基因相对于其他H链tRNA基因约高25倍的转录速率，以及L链tRNA基因相对于H链tRNA基因高10 - 16倍的转录速率，并未反映在相应tRNA的稳态水平或形成速率上。将各个tRNA的稳态水平与从DNA序列和各种多肽的合成速率确定的蛋白质合成密码子使用情况进行比较，未发现这两个参数之间有任何显著相关性。
