The Association of Three Subunits with Yeast RNA Polymerase Is Stabilized by A14 (*)
The Association of Three Subunits with Yeast RNA Polymerase Is Stabilized by A14 (*)
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A14 稳定了三个亚基与酵母 RNA 聚合酶的结合 (*)
DOI：
10.1074/jbc.270.22.13534
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
C. Carles
中科院分区：
文献类型：
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作者：
Amke Smid;M. Riva;Franoise Bouet;A. Sentenac;C. Carles
酿酒酵母RNA聚合酶I由14个亚基组成。除了编码A14的RPA14基因外，所有相应的基因都已被克隆。A14是该酶的一种特殊多肽。我们报道了RPA14的克隆和鉴定。用反相高效液相色谱法从RNA聚合酶I的其他亚基中分离出A14多肽，并用蛋白酶K消化，根据其中一种多肽的氨基酸序列合成简并寡核苷酸，并用于从酵母亚基因组DNA文库中分离RPA14基因。RPA14是一个单拷贝基因，位于第四染色体上，其两侧是CYP1和HOM2。RPA14的破坏不是致命的，但在37℃和38℃时，rpa14：：URA3突变株的生长受到损害。RNA聚合酶I是从rpa14：：URA3菌株中纯化的。经过两步纯化后，该酶不含A14、ABC23和A43亚基。这种形式的酶在非特异性体外转录试验中不具有活性。这些结果表明，A14是RNA聚合酶I的一个真正的亚基，并提示A14在亚基的稳定性中发挥作用。
RNA polymerase I of Saccharomyces cerevisiae is composed of 14 subunits. All of the corresponding genes have been cloned with the exception of the RPA14 gene encoding A14, a specific polypeptide of this enzyme. We report the cloning and the characterization of RPA14. The A14 polypeptide was separated from the other RNA polymerase I subunits by reverse-phase high pressure liquid chromatography and digested with proteinase K. Based on the amino acid sequence of one of the resulting peptides, a degenerate oligonucleotide was synthesized and used to isolate the RPA14 gene from a yeast subgenomic DNA library. RPA14 is a single copy gene that maps to chromosome IV and is flanked by CYP1 and HOM2. Disruption of RPA14 is not lethal, but growth of the rpa14::URA3 mutant strain is impaired at 37 and 38°C. RNA polymerase I was purified from the rpa14::URA3 strain. After two purification steps, the enzyme did not contain the subunits A14, ABC23, and A43. This form of the enzyme was not active in a nonspecific in vitro transcription assay. These results demonstrate that A14 is a genuine subunit of RNA polymerase I and suggest that A14 plays a role in the stability of a subgroup of subunits.