In vitro synthesis of transfer RNA. I. Purification of required components.
In vitro synthesis of transfer RNA. I. Purification of required components.
复制标题
转移RNA的体外合成。
DOI：
10.1016/s0021-9258(19)41057-0
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发表时间：
1975
期刊：
影响因子：
--
通讯作者：
M. Gefter
中科院分区：
文献类型：
--
作者：
E. Bikoff;M. Gefter
我们已经描述了一种体外系统，其中活性su+III tRNATyr从phi 80 psu ++III DNA模板合成。使用这个系统，我们已经确定了合成tRNA所需的四个基本组成部分。第一种是依赖于DNA的RNA聚合酶。已经表明，DNA依赖性RNA聚合酶的粗制剂合成与体内分离的类似的su++III tRNATyr前体，并且该制剂能够支持高水平的tRNA合成。纯化的DNA依赖的RNA聚合酶，苏++III tRNATyr前体没有观察到作为转录产物和tRNA合成低于detetable水平。在此基础上，确定了tRNA合成的第二个必需组分。该级分，命名为级分V，与纯化的RNA聚合酶组合，催化前体tRNA的合成。第三种成分是核糖核酸酶（RNase P III），其特异性催化去除存在于tRNA前体3'末端的额外核苷酸。在没有这一部分，在体外合成的苏++III tRNATyr是略大于4 S，并包含额外的核苷酸超出了正常的-CCAOH 3末端的成熟tRNA。所需的第四种必需组分是含有RNase P的级分，RNase P是一种先前鉴定的内切核酸酶，其特异性催化tRNA前体上存在的5'额外核苷酸的去除。
We have described an in vitro system in which active su+III tRNATyr is synthesized from a phi80psu++III DNA template. Using this system, we have identified four essential components that are required for synthesis of tRNA. The first of these is DNA-dependent RNA polymerase. It has been shown that a crude preparation of DNA-dependent RNA polymerase synthesizes su++III tRNATyr precursor similar to that which has been isolated in vivo, and that this preparation is capable of supporting high levels of tRNA synthesis. With purified DNA-dependent RNA polymerase, the su++III tRNATyr precursor was not observed as a transcription product and tRNA synthesis was below detetable levels. On this basis, a second essential component for tRNA synthesis was identified. This fraction, designated Fraction V, in combination with purified RNA polymerase, catalyzes the synthesis of precursor tRNA. The third component is a ribonuclease (RNase P III), which specifically catalyzes the removal of the extra nucleotides present at the 3' terminus of the tRNA precursor. In the absence of this fraction, the in vitro synthesized su++III tRNATyr is slightly larger than 4 S and contains additional nucleotides beyond the normal --CCAOH 3 terminus of the mature tRNA. The fourth essential component required is a fraction containing RNase P, a previously identified endonuclease which specifically catalyzes the removal of the 5' extra nucleotides present on tRNA precursors.