PHYSIOLOGICAL GENETICS OF EUKARYOTIC RNA POLYMERASE III

酵母RET1基因编码130kd的RNA聚合酶III亚基， 将受到区域突变，随后将进行筛查 改变了RNA聚合酶III的活性。RET1蛋白属于一种 超家族，包括所有核和子中第二大亚单位 细菌聚合酶。所有家庭成员都有12种蛋白质 序列基序，在其中四个基序中，我们将带来氨基酸 替换。特别令人感兴趣的是由77个氨基酸组成的序列 部分与β亚基的Rif区部分同源的长度 E.ColiRNA聚合酶。许多残基中的简单氨基酸取代 整个Rif区域和相应的RET序列改变 RNA聚合酶对终止信号的响应。通过对这些进行排序 含RET1突变的Pol III的活性研究 突变的亚基，我们试图推断该蛋白区域在 转录延伸和终止。 许多阻止转录过程中特定步骤的RET1突变是 致命的。含有致命性RET1突变蛋白的POL III只能被回收 来自杂合子细胞。从他们那里获得一种纯净的突变体提取物 转录研究，我们开发了一种基于 基于表位标记和对野生型POL III的选择性移除。

The yeast RET1 gene, which encodes a 130 kd subunit of RNA polymerase III, will be subjected to regional mutagenesis, with subsequent screening for altered RNA polymerase III activity. The RET1 protein belongs to a superfamily that includes the second largest subunit of all nuclear and bacterial polymerases. Common to all family members are 12 protein sequence motifs, in four of which we will bring about amino acid substitutions. Of particular interest is a sequence 77 amino acids in length that is in part homologous to the Rif region of the Beta subunit of E. coli RNA polymerase. Simple amino acid substitutions in many residues throughout the Rif region and the corresponding RET sequences change the response of RNA polymerase to termination signals. By sequencing these RET1 mutations and characterizing the activity of Pol III containing the mutant subunits, we seek to infer the function of this protein region in transcription elongation and termination. Many of the RET1 mutations that block a specific step in transcription are lethal. Pol III with a lethal RET1 mutant protein can only be recovered from heterozygous cells. To obtain from them a pure mutant extract for transcription studies, we have developed an immunoselection method based upon epitope tagging and selective removal of the wild type Pol III.