FORMATION OF AN RNA PRIMER FOR INITIATION OF REPLICATION OF COLE1 DNA BY RIBONUCLEASE-H
FORMATION OF AN RNA PRIMER FOR INITIATION OF REPLICATION OF COLE1 DNA BY RIBONUCLEASE-H
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DOI：
10.1073/pnas.77.5.2450
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发表时间：
1980-01-01
期刊：
影响因子：
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通讯作者：
TOMIZAWA, J
中科院分区：
文献类型：
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作者：
ITOH, T;TOMIZAWA, J
一种质粒，由含有[大肠杆菌]质粒ColE1复制起始的812碱基对片段和含有. β的1240碱基对片段组成构建-内酰胺酶基因。质粒DNA在体外有3个主要的转录起始位点。一个位于ColE1片段555核苷酸上游。大多数来自这个位点的转录延伸到起源之外；一些转录本在其3”部分自发地与模板形成杂交体。RNase H切割这些转录本在起始区产生3‘’末端。当DNA聚合酶I与RNA聚合酶和RNase H一起参与反应时，将dAMP或dCMP直接添加到裂解的RNA分子上，其中大多数保留完整的5”端。在被切割的RNA上加入一个脱氧核糖核苷酸可以看作是ColE1 DNA合成的第一步。一旦作为引物，RNA就会被RNase H从产物中清除。
A plasmid that consists of an 812-base-pair segment containing the replication origin of [Escherichia coli] plasmid ColE1 and of a 1240-base-pair segment containing a .beta.-lactamase gene was constructed. The plasmid DNA has 3 principal sites where transcription is initiated in vitro. One is located in the ColE1 segment 555 nucleotides upstream from the origin. Most transcription from this site extends past the origin; some of the transcripts form hybrids spontaneously with the template at their 3'' portions. Cleavage of these transcripts by RNase H generates 3'' termini at the origin region. When DNA polymerase I is included in the reaction along with RNA polymerase and RNase H, dAMP or dCMP is added directly onto the cleaved RNA molecules, most of which retain the intact 5'' terminus. The addition of a deoxyribonucleotide to the cleaved RNA can be regarded as the 1st step of ColE1 DNA synthesis. Once it served as a primer, the RNA is eliminated from the product by RNase H.