Nishikura K, Kurjan J, Hall B D, De Robertis E M
EMBO J. 1982;1(2):263-8. doi: 10.1002/j.1460-2075.1982.tb01157.x.
We analyzed the effect of 18 single nucleotide changes on the processing of the transcripts produced by cloned yeast tRNATyr genes after microinjection into the nucleus of living Xenopus oocytes. The processing step most easily blocked by mutation is the early maturation of the 5' and 3' termini of the tRNATyr primary transcript, involving removal of 5'-leader and 3'-trailer sequences and CCA addition. The enzymes seem to recognize the whole tRNA cloverleaf structure since mutations in all regions of the molecule can stop processing. Mutations that affect splicing of the 92-nucleotide precursor (which has mature ends but still contains the intervening sequence, and is the normal substrate for the splicing enzymes), are located in the vicinity of the intervening sequence. Base modification enzymes that add pseudouridine, 1-methyladenosine and 5-methylcytosine appear rather insensitive to changes in secondary and tertiary structure of early transcripts in the 16 mutants examined. These enzymes may recognize only limited regions of the precursor RNA. RNA polymerase III behaves as if able to count the number of Us added before termination; and aberrant termination products in two mutants suggest that the secondary structure of the nascent transcript can be very imortant in eukaryotic transcription termination.
我们分析了18个单核苷酸变化对克隆的酵母tRNATyr基因转录本加工过程的影响，这些转录本在显微注射到非洲爪蟾卵母细胞的细胞核后产生。最容易因突变而受阻的加工步骤是tRNATyr初级转录本5′和3′末端的早期成熟，包括去除5′前导序列和3′拖尾序列以及添加CCA。这些酶似乎能识别整个tRNA三叶草结构，因为分子所有区域的突变都能阻止加工过程。影响92个核苷酸前体（其末端已成熟，但仍含有间隔序列，是剪接酶的正常底物）剪接的突变，位于间隔序列附近。在所检测的16个突变体中，添加假尿苷、1-甲基腺苷和5-甲基胞嘧啶的碱基修饰酶对早期转录本二级和三级结构的变化相当不敏感。这些酶可能只识别前体RNA的有限区域。RNA聚合酶III的行为似乎表明它能够在终止前计算添加的U的数量；两个突变体中的异常终止产物表明，新生转录本的二级结构在真核生物转录终止中可能非常重要。
