PHYSIOLOGICAL GENETICS OF EUKARYOTIC RNA POLYMERASE III

酵母RET 1基因编码RNA聚合酶III的130 kd亚基， 将进行区域诱变，随后筛选 改变RNA聚合酶III活性。 RET1蛋白属于 一个包括所有核和 细菌聚合酶 所有家族成员共有12种蛋白质 序列基序，在其中四个，我们将带来氨基酸 替代品 特别令人感兴趣的是一个77个氨基酸的序列， 长度，该长度部分同源于以下的β亚基的Rif区域： E. coli RNA聚合酶。 许多残基中的简单氨基酸取代 在整个Rif区域和相应的RET序列中， RNA聚合酶对终止信号的反应。 通过对这些基因进行测序 RET1突变和表征含有RET1突变的Pol III的活性。 突变的亚基，我们试图推断这个蛋白质区域的功能， 转录延长和终止。 许多阻止转录中特定步骤的RET 1突变是 致命的 具有致死性RET 1突变蛋白的Pol III仅可回收 从杂合细胞中分离出来。 从他们身上获得纯的突变体提取物， 转录研究，我们已经开发了一种免疫选择方法， 在表位标记和选择性去除野生型Pol III后。

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.