The archaeal RNA polymerase subunit P and the eukaryotic polymerase subunit Rpb12 are interchangeable in vivo and in vitro
The archaeal RNA polymerase subunit P and the eukaryotic polymerase subunit Rpb12 are interchangeable in vivo and in vitro
复制标题
古细菌 RNA 聚合酶亚基 P 和真核聚合酶亚基 Rpb12 在体内和体外可互换
作者：
C. Reich;M. Zeller;P. Milkereit;W. Hausner;P. Cramer;H. Tschochner;M. Thomm
所有三种真核RNA聚合酶的通用亚基Rpb12和古生菌酶的亚基P在N末端的锌带和C末端的一些高度保守的残基上显示出序列相似性。我们在这里报道了在酵母强启动子的控制下的古生菌P亚基可以补充Rpb12缺失突变体的致死表型，并且在古生菌RNA聚合酶的重组过程中，酵母的Rpb12亚基可以在功能上取代P亚基。ΔP酶不能形成稳定的开放复合体，但可以有效地将二核苷酸延伸到预熔化的模板上或延伸支架上的RNA.这表明P亚基直接或间接地参与了启动子的开放。ΔP酶的活性可以通过在转录反应中加入Rpb12或P亚基来挽救。锌带上半胱氨酸残基的突变在几种检测中会削弱酶的活性，这种突变形式的P在转录反应中很快被野生型P取代。N-末端保守的锌带似乎对整个亚基与其他RNA聚合酶亚基的正确相互作用是重要的，一个17个氨基酸的C-端肽足以支持体外所有基本的RNA聚合酶功能。
The general subunit of all three eukaryotic RNA polymerases, Rpb12, and subunit P of the archaeal enzyme show sequence similarities in their N‐terminal zinc ribbon and some highly conserved residues in the C‐terminus. We report here that archaeal subunit P under the control of a strong yeast promoter could complement the lethal phenotype of a RPB12 deletion mutant and that subunit Rpb12 from yeast can functionally replace subunit P during reconstitution of the archaeal RNA polymerase. The ΔP enzyme is unable to form stable open complexes, but can efficiently extend a dinucleotide on a premelted template or RNA on an elongation scaffold. This suggests that subunit P is directly or indirectly involved in promoter opening. The activity of the ΔP enzyme can be rescued by the addition of Rpb12 or subunit P to transcription reactions. Mutation of cysteine residues in the zinc ribbon impair the activity of the enzyme in several assays and this mutated form of P is rapidly replaced by wild‐type P in transcription reactions. The conserved zinc ribbon in the N‐terminus seems to be important for proper interaction of the complete subunit with other RNA polymerase subunits and a 17‐amino‐acid C‐terminal peptide is sufficient to support all basic RNA polymerase functions in vitro.